Compounds Having Activity in Increasing Ion Transport by Mutant-CFTR and Uses Thereof

ABSTRACT

The invention provides compositions, pharmaceutical preparations and methods for increasing activity (e.g., ion transport) of the mutant cystic fibrosis transmembrane conductance regulator protein (mutant-CFTR), e.g., DF508 CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR, that are useful for the treatment of cystic fibrosis (CF). The compositions and pharmaceutical preparations of the invention may comprise one or more phenylglycine-containing compounds or sulfonamide-containing compounds of the invention, or an analog or derivative thereof.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant nos.HL73856, EB00415, HL59198, EY13574, and DK35124 awarded by the NationalInstitutes of Health. The government may have certain rights in thisinvention.

Work on this invention was also supported by grants from the CysticFibrosis Foundation and/or from Cystic Fibrosis Foundation Therapeutics.

BACKGROUND OF THE INVENTION

The cystic fibrosis transmembrane conductance regulator protein (CFTR)is a cAMP-activated chloride (Cl⁻) channel expressed in epithelial cellsin mammalian airways, intestine, pancreas and testis. CFTR is thechloride-channel responsible for cAMP-mediated Cl⁺ secretion. Hormones,such as a β-adrenergic agonist, or toxins, such as cholera toxin, leadto an increase in cAMP, activation of cAMP-dependent protein kinase, andphosphorylation of the CFTR Cl⁻ channel, which causes the channel toopen. An increase in the concentration of Ca²⁴⁺ in a cell can alsoactivate different apical membrane channels. Phosphorylation by proteinkinase C can either open or shut Cl⁻ channels in the apical membrane.CFTR is predominantly located in epithelia where it provides a pathwayfor the movement of Cl⁻ ions across the apical membrane and a key pointat which to regulate the rate of transepithelial salt and watertransport. CFTR chloride channel function is associated with a widespectrum of disease, including cystic fibrosis (CF) and with some formsof male infertility, polycystic kidney disease and secretory diarrhea.

The hereditary lethal disease CF is caused by mutations in the geneencoding the CFTR protein, a cAMP-activated Cl⁻ channel expressed inairway, intestinal, pancreatic, and other secretory and absorptiveepithelia. The principal clinical problem in CF is recurrent lunginfections resulting in progressive deterioration in lung function. Themost common CFTR mutation, deletion of phenylalanine-508 (ΔF508-CFTR),is present in at least one allele in about 90% of CF patients (Egan etal., (2004) Science 304:600-602). ΔF508-CFTR causes Cl⁺ impermeabilitybecause it is not processed correctly, causing it to be retained at theendoplasmic reticulum (rather than the plasma membrane). ΔF508-CFTR alsohas reduced intrinsic Cl⁺ conductance relative to wild type CFTR.

Strategies have been investigated to correct the defects in ΔF508-CFTRcellular processing and intrinsic function in cells. Cell growth at lowtemperature (<30° C.) (Denning et al., (1992) Nature 358, 761-764) orwith high concentrations of chemical chaperones such as glycerol (Satoet al., (1996) J. Biol. Chem. 271, 635-638; Brown, et al., (1996) CellStress & Chaperones 1, 117-125) corrects partially defective ΔF508-CFTRcellular processing by a mechanism that may involve improved proteinfolding and stability (Sharma et al., (2001) J. Biol. Chem. 276,8942-8950). A sustained increase in intracellular calcium concentrationby thapsigargin also corrects defective ΔF508-CFTR processing (Egan etal., (2002) Nature Med. 8, 485-492), possibly by interfering withinteractions with molecular chaperones. Compounds like phenylbutryatefacilitate ΔF508-CFTR cellular processing by altering chaperone functionand/or transcriptional enhancement (Rubenstein et al., (2000) Am. J.Physiol. 278, C259-C267; Kang et al., (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 838-843). Although these approaches provide insight intomechanisms of ΔF508-CFTR retention at the endoplasmic reticulum, theyprobably do not offer clinically-useful therapies.

ΔF508-CFTR has significantly impaired channel activity even when presentat the cell plasma membrane (Dalemans et al., (1991) Nature 354,526-528). Cell-attached patch-clamp measurements showed reducedΔF508-CFTR open channel probability and prolonged closed times even withmaximal cAMP stimulation (Haws et al., (1996) Am. J. Physiol. 270,C1544-C1555; Hwang et al., (1997) Am. J. Physiol. 273, C988-C998).Patch-clamp measurements in excised membranes indicated 7-fold reducedΔF508-CFTR activation after phosphorylation compared to wildtype CFTR.Relatively high concentrations of the flavone genistein (>50 μM, Hwang,et al., (1997) Am. J. Physiol. 273, C988-C998; Wang et al., (2000) J.Physiol. 524, 637-638) or the xanthine isobutylmethylxanthine (>1 mM,Drumm et al., (1991) Science 254, 1797-1799) in combination with cAMPagonists increase ΔF508-CFTR channel activity. Again, these studies havenot offered any clinically useful therapies.

There is accordingly still a need for compounds that can activate mutantCFTR, e.g., ΔF508-CTFR G551D-CFTR, or G1349D-CFTR, and methods of usingsuch compounds for the study and treatment of CF and the treatment andcontrol of other secretory disorders. The present invention addressesthese needs, as well as others.

SUMMARY OF THE INVENTION

The invention provides compositions, pharmaceutical preparations andmethods for increasing activity (e.g., ion transport) of a mutant-cysticfibrosis transmembrane conductance regulator protein (e.g., ΔF508 CFTR,G551D-CFTR, G1349D-CFTR, or D1152H-CFTR) that are useful for thetreatment of cystic fibrosis (CF). The compositions and pharmaceuticalpreparations of the invention may comprise one or morephenylglycine-containing compounds or sulfonamide-containing compoundsof the invention, or an analog or derivative thereof.

The invention provides for a pharmaceutical composition comprising acompound of formula (I):

where n R₁ is independently chosen from a substituted or unsubstitutedphenyl group or a substituted or unsubstituted heteroaromatic group, ora cyclic or acyclic alkyl group; R₂ is independently chosen form ahydrogen, a alkyl group, an ether group, a halogen, or a perfluoroalkylgroup; R₃ is independently chosen from a hydrogen or an alkyl group, andR₄ is independently chosen from a substituted or unsubstitutedheteroaromatic group, or a alkanoyl-amine group; or a pharmaceuticallyacceptable derivative thereof, as an individual stereoisomer or amixture thereof; or a pharmaceutically acceptable salt thereof. In oneembodiment, the composition further includes at least one of apharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient and a pharmaceuticallyacceptable adjuvant. In another embodiment the composition does notcontain detectable dimethyl sulfoxide. In preferred embodiments, thecompound is chosen from:2-[(2-1H-Indol-3-yl-acetyl)-methyl-amino]-N-(4-isopropyl-phenyl)-2-phenyl-acetamide;2-[(2-1H-Indol-3-yl-acetyl)-methyl-amino]-N-(4-isopropyl-phenyl)-2-(4-methoxy-phenyl)-acetamide;2-[(2-1H-Indol-3-yl-acetyl)-methyl-amino]-N-(4-methoxy-phenyl)-2-phenyl-acetamide;2-[(2-1H-Indol-3-yl-acetyl)-methyl-amino]-2,N-bis-(4-methoxy-phenyl)-acetamide;N-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-2-(2-1H-indol-2-yl-acetylamino)-2-p-tolyl-acetamide;N-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)-2-[(2-1H-indol-3-yl-acetyl)-methyl-amino]-2-(4-methoxy-phenyl)-acetamide;2-(2-1H-Indol-3-yl-acetylamino)-N-(4-isopropyl-phenyl)-2-phenyl-acetamide;N-Benzo[1,3]dioxol-5-yl-2-[(2-1H-indol-3-yl-acetyl)-methyl-amino]-2-p-tolyl-acetamide;or2-[(2-Acetylamino-acetyl)-methyl-amino]-N-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-2-phenyl-acetamide.

In one embodiment R₁ is chosen from a phenyl group substituted by ahydrogen, a methyl group, an isobutanyl group, or a methoxyl group. Inanother embodiment, R₂ is chosen from a hydrogen, a methyl group, or amethoxyl group. In yet another embodiment R₃ is chosen from a hydrogenor a methyl group. In yet another embodiment, R₄ is chosen from anindole group or an alkanoylamino group.

In another embodiment of particular interest, R₁ is independently chosenfrom a substituted or unsubstituted heteroaromatic group; R₂ isindependently chosen form a hydrogen, a alkyl group, or an ether group;R₃ is independently chosen from a hydrogen or an alkyl group, and R₄ isindependently chosen from a substituted or unsubstituted heteroaromaticgroup, or a alkanoylamino group. In one embodiment, R₆ is a2,3-dihydro-benzo[1,4]dioxine group. In another embodiment, R₂ is chosenfrom a hydrogen, a methyl group, or a methoxyl group. In yet anotherembodiment, R₃ is chosen from a hydrogen or a methyl group. In yetanother embodiment, R₄ is chosen from an indole group or an acetylaminogroup.

The invention also provides for a pharmaceutical composition comprisinga compound of formula (II):

wherein R₁ is independently chosen form a hydrogen, an alkyl groupunsubstituted or substituted by an alkoxy group; R₂ is independentlychosen from a hydrogen or a substituted or unsubstituted phenyl group;R₃ is independently selected from an alkyl group unsubstituted orsubstituted by an alkoxy group, a substituted or unsubstitutedhydrocarbon cyclic ring group, or a substituted or unsubstitutedheterocyclic ring; or a pharmaceutically acceptable derivative thereof,as an individual stereoisomer or a mixture thereof; or apharmaceutically acceptable salt thereof. In some embodiments thecomposition further includes at least one of a pharmaceuticallyacceptable carrier, a pharmaceutically acceptable diluent, apharmaceutically acceptable excipient and a pharmaceutically acceptableadjuvant. In one embodiment, the composition does not contain detectabledimethyl sulfoxide. In another embodiment, R₁ is chosen from a hydrogen,a phenyl group, a 3-fluorophenyl, a 3-methylphenyl group, a2-methylphenyl group, a 2,6-dimethylphenyl group, or a 2-ethoxyphenylgroup. In another embodiment, R₂ is chosen from a methyl group, an ethylgroup, or a propylene group. In yet another embodiment, R₃ is chosenfrom a butyl group, a propylene group, an isopentyl group, amethoxy-propane group, a cyclopentyl group, a cylcohexyl group, a2-methyl-furan group, or a 2-methyl-tetrahydro-furan group.

In an embodiment of particular interest the compound of formula (II) isa compound of formula (IIa):

wherein R₄ is a substituted or unsubstituted heterocycloalkyl groupcontaining a nitrogen atom, wherein the heterocycloalkyl group is linkedto the sulfur atom by the nitrogen atom of the heterocycloalkyl group, asubstituted or unsubstituted heterocyclic group; R₃ is independentlyselected from an alkyl group unsubstituted or substituted by an alkoxygroup, a substituted or unsubstituted hydrocarbon cyclic ring group, ora substituted or unsubstituted heterocyclic ring. In an embodiment, R₄is chosen from a 1,4-Dioxa-8-aza-spiro[4.5]decane group or a2,3-Dihydro-1H-indole group. In another embodiment, R₃ is chosen from abutyl group, a propylene group, an isopentyl group, a 3-methoxy-propylgroup, a cyclopentyl group, a cylcohexyl group, a 2-methyl-furan group,or a 2-methyl-tetrahydrofuran group. In preferred embodiments, thecompound is chosen from:6-[(2-Ethoxy-phenyl)-methyl-sulfamoyl]-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid allylamide;6-(Ethyl-phenyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid(3-methoxy-propyl)-amide;6-(Methyl-m-tolyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid (pyridin-2-ylmethyl)-amide;6-(Methyl-m-tolyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid (2-cyclohex-1-enyl-ethyl)-amide;6-(1,4-Dioxa-8-aza-spiro[4.5]decane-8-sulfonyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid (3-methyl-butyl)-amide;6-[Ethyl-(4-fluoro-phenyl)-sulfamoyl]-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid cyclopentylamide;6-(Methyl-o-tolyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid (3-methyl-butyl)-amide;6-[(2,6-Dimethyl-phenyl)-methyl-sulfamoyl]-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid butylamide;6-(Allyl-phenyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid(furan-2-ylmethyl)-amide;6-[Ethyl-(4-fluoro-phenyl)-sulfamoyl]-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid (tetrahydro-furan-2-ylmethyl)-amide;6-(Methyl-m-tolyl-sulfamoyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid sec-butylamide; or6-(2,3-Dihydro-indole-1-sulfonyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid cyclohexylamide.

The invention also provides for a method of treating a subject having acondition associated with mutant-CFTR, the method includingadministering to the subject a therapeutically effective amount of acompound selected from the compounds of the present invention. In someembodiments, the condition is cystic fibrosis. In some embodiments thesubject, after treatment, has a decrease in mucous or bacterial titer intheir lungs, a decrease in coughing or wheezing, a decrease inpancreatic insufficiency, or a decrease in electrolyte levels in theirsweat. In some embodiments the subject is a non-human animal. Inembodiments of particular interest the animal is a mammal. In someembodiments the mutant-CFTR is ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, orD1152H-CFTR.

The invention also provides for a method of increasing ion permeabilityof a cell producing a mutant-CFTR protein, the method includingcontacting the cell with a compound in an amount effective to increaseion permeability of said cell, wherein the compound is selected from thecompounds of the present invention. In some embodiments the cellcontains a recombinant expression cassette that encodes said mutant-CFTRprotein. In other embodiments the cell contains a genome that encodessaid mutant-CFTR protein. In yet other embodiments the ion permeabilityincreases an ion transporting activity that increases a rate oftransport of ions across the plasma membrane of said cell. In yet otherembodiments the mutant-CFTR is ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, orD1152H-CFTR.

These and other objects and advantages of the invention will be apparentfrom the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing drawings, which are for illustrative purposes only.

FIG. 1 shows the details of identification of the subject compounds.Panel A is a schematic representation of a high-throughput screeningprocedure used in the subject methods. Cells co-expressing mutant-CFTRand the halide-sensitive fluorescent protein YFP-H148Q/I152L were grownfor 24 h at 27° C. (to give plasma membrane mutant-CFTR expression).After washing, test compounds (2.5 μM) and forskolin (20 μM) were added,and I⁻ influx was assayed from the time course of YFP-H148Q/I152Lfluorescence after adding I⁻ to the external solution. Panel B shows theoriginal traces showing quenching of cellular YFP fluorescence by I⁻addition with saline alone, and after additions of forskolin (20 μM)alone, or forskolin plus genistein (50 μM), compound S-1 (2.5 μM) orcompound P-1 (2.5 μM).

FIG. 2 shows the synthesis and structure activity analysis of thesubject compound. Panel A shows the structures of an exemplaryphenylglycine containing compound (denoted as P-1) and an exemplarysulfonamide containing compound (denoted as S-1). Panel B, top portion,shows the synthesis of the phenylglycine containing compound P-1.Conditions: a. p-isopropylaniline, EDCI, cat. (catalytic amount) DMAP,CH₂Cl₂, 22° C., 2 h, yield 92%; b. TFA, 22° C., 15 min, 98%; c.indole-3-acetic acid, EDCI, cat. DMAP, CH₂Cl₂, 22° C., 2 h, 92%. PanelA, bottom portion, shows the synthesis of the sulfonamide containingcompound S-3. Conditions: d. diethyl ethoxymethylene-malonate, 140° C.,1 h, 95%; e. cat. p-chlorobenzoic acid. Ph₂O, 250° C., 45%; f.o-methoxybenzyl-amine, neat, 180° C., 35%. Panel C shows the conclusionsfrom structure-activity relationship analysis of the phenylglycinecontaining compounds and the sulfonamide containing compounds.

FIG. 3 provides dose response analysis of the subject compounds. Panel Ais a graph showing I⁻ influx rates (d[I⁻]/dt) for phenylglycinecontaining compounds. Panel B is a graph showing I⁻ influx rates(d[I⁻]/dt) for sulfonamide containing compounds. Panel C is a graphshowing I⁻ influx rates (d[I⁻]/dt) for the indicated compounds (mean±SE,n=4), including the tetrahydrobenzothiophene ΔF508_(act)-02 (Yang et.al., JBC 278:35079-35085 (2003)).

FIG. 4 provides graphs showing CFTR-mediated chloride currents measuredin FRT cells expressing ΔF508-CFTR for the phenylglycine containingcompound P-1 (Panel A, left), the sulfonamide containing compound S-1(Panel A, right) in the presence of forskolin, and the averagedose-responses for the compounds, with genistein data shown forcomparison (SE, n=4) (Panel B).

FIG. 5 provides the results of Ussing chamber experiments. Panel Aprovides representative traces showing potentiation of the response ofΔF508-CFTR to forskolin in the absence (upper graph) or presence (lowergraph) of a phenylglycine containing compound (P-1). Panel B of FIG. 5shows a summary of similar experiments for P-1 and a sulfonamidecontaining compound (S-1) which show significant increase in currentinduced by low concentrations of forskolin.

FIG. 6 shows the specificity of the subject compounds. Panel A showsintracellular cAMP concentration after forskolin addition with andwithout compounds P-1 and S-1 (2 μM). Panel B shows MDR-1 activity shownas rhodamine 123 accumulation in multidrug sensitive (9HTEo-) andmultidrug resistant (9HTEo-/Dx) cells. Significant accumulation wasfound in 9HTEo-/Dx cells for verapamil (100 μM) but not for compoundsP-1 and S-1 (5 μM). Panel C shows activation of Cl⁺ current by apicalUTP in polarized human bronchial epithelia. Pretreatment with ΔF508-CFTRactivators (2 μM) did not affect the maximum current or time-course ofthe UTP response.

FIG. 7 provides graphs illustrating representative examples ofpotentiator effects as detected by patch-clamp analysis. Panel A showscell-attached patch-clamp recordings of ΔF508-CFTR channel activity inthe presence of forskolin (20 μM) (top portion) and after addition ofthe phenylglycine containing compound P-1 (100 nM) or the sulfonamidecontaining compound S-1 (bottom portion, 100 nM). Panel B is a series ofgraphs summarizing the average averaged channel open probabilities (Po)(left), mean closed time (T_(c)) (middle), and mean open time (T_(o))(right) in the presence of forskolin alone or in combination withindicated compounds from the data of Panel A.

FIG. 8 is a set of graphs showing stimulation of Cl-secretion in CFhuman airway epithelial cells. Panel A shows ΔF508-CFTR activation innasal epithelial cells from a ΔF508-CFTR homozygous subject afteraddition of compound P-1 (left panel, bottom portion), compound S-1(right panel, bottom portion) in the presence of forskolin followingaddition of amiloride to block epithelial sodium channels, or genisteinat either 37° C. (left panel, top portion) or 27° C. (right panel, topportion). Panel B shows G551D-CFTR activation in nasal epithelial cellsfrom a G551D-CFTR homozygous subject after addition of compound P-1.Panel C shows D1152H-CFTR activation in nasal epithelial cells from aD1152H-CFTR homozygous subject after addition of compound P-1.

FIG. 9 shows results of activation of G551D- and G1349D-CFTR mutants.Panels A and B show CFTR-mediated chloride currents measured inepithelial cells expressing either G551D-CFTR (Panel A) or G1349D-CFTR(Panel B) in response to the addition of either the phenylglycinecontaining compound P-1 (bottom portion of each panel) or genestein (topportion of each panel) in the presence of forskolin. Panels C and D areresults of dose-response curves (SE, n=4) for compound P-1 and genisteinfor activation of G551D-CFTR (Panel C) and G1349D-CFTR (Panel D).

FIG. 10 is a set of graphs showing CFTR-mediated chloride currentsmeasured in nasal polyp epithelial cells from a CF patient withG551D-CFTR mutation in response to the addition of either thephenylglycine containing compound P-1 (right panel) or genestein (leftpanel) in the presence of forskolin following addition of amiloride toblock epithelial sodium channels.

FIG. 11 shows liquid chromatography/mass spectrometry analysis ofmicrosomal metabolites of compounds P-1 and S-3, and ratpharmacokinetics. Panel A shows results of the liquidchromatography/mass spectrometry analysis. Microsomes were incubatedwith compounds P-1 or S-3 (each 10 μM) in the absence (control) orpresence of NADPH for 1 hour at 37° C. HPLC chromatograms at 256 nm forcontrol (left) and NADPH (right) samples, and corresponding ion currentchromatograms for positive ion electrospray mass spectrometry forindicated m/z (middle). M1, metabolite 1; M2, metabolite 2. Panel Bshows pharmacokinetic analysis. The left panel shows the HPLCchromatogram of compounds P-1 and S-3 demonstrating assay sensitivity tobetter than 50 nM. The right panel shows the pharmacokinetics ofcompounds P-1 (open circles) and S-3 (closed circles) after 5 mg/Kgintravenous bolous injection (mean±SE, n=3-4 rats).

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It should be noted that, as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds, and reference to “thecell” includes reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application, and areincorporated herein by reference. Nothing herein is to be construed asan admission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication datesthat may need to be independently confirmed.

The definitions used herein are provided for reason of clarity, andshould not be considered as limiting. The technical and scientific termsused herein are intended to have the same meaning as commonly understoodby those of ordinary skill in the art to which the invention pertains.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions, pharmaceutical preparations andmethods for activation of mutant cystic fibrosis transmembraneconductance regulator protein (e.g., ΔF508-CFTR, G551D-CFTR,G1349D-CFTR, or D1152H-CFTR) that are useful for the study and treatmentof cystic fibrosis (CF). The invention also features methods of use ofsuch compositions in increasing activity of mutant CFTR in a cell, e.g.,by increasing ion transport by mutant CFTR.

In one embodiment, the compositions and pharmaceutical preparations ofthe invention may comprise one or more compounds, which compounds can bea phenylglycine containing compound, or an analog or derivative thereof,and a sulfonamide containing compound, or an analog or derivativethereof. The compositions and pharmaceutical preparations of theinvention may additionally comprise one or more pharmaceuticallyacceptable carriers, excipients and/or adjuvants.

The invention provides methods of increasing ion transport in amutant-CFTR, e.g., ΔF508-CFTR G551D-CFTR, G1349D-CFTR, or D1152H-CFTR,in a cell by contacting the cell with an effective amount of one or moreof the compounds set forth above. In other embodiments, the inventionalso provides a method of treating a patient suffering from amutant-CFTR-mediated disease or condition, for example CF, byadministering to the patient an efficacious amount of one or more of thecompounds set forth above. Kits for use in the subject methods are alsoprovided.

In one aspect of particular interest, the invention is based on thediscovery of a genus of phenylglycine containing compounds that increaseion transport by mutant-CFTR with high affinity.

In another aspect of particular interest, the invention is based on thediscovery of a genus of sulfonamide containing compounds that increaseion transport by mutant-CFTR with high affinity.

In describing the invention, the structure of the compounds of theinvention will be described first. Then, pharmaceutical formulationscontaining the compounds will be discussed, followed by a description oftheir methods of use.

DEFINITIONS

A “mutant cystic fibrosis transmembrane conductance regulator protein”,or “mutant-CFTR” is the protein that results from a mutation, e.g.,deletion mutation, insertion mutation, or point (substitution) mutationof the CFTR gene product relative to wildtype. As used herein a “mutantcystic fibrosis transmembrane conductance regulator protein”, or“mutant-CFTR” is dysfunctional as compared to a functional (e.g.,wildtype) CFTR where the dysfunction can encompass one or more of thefollowing: (i) aberrant CFTR production (e.g., at the level oftranscription or translation); (ii) aberrant folding and/or trafficking;(iii) abnormal regulation of conductance; (iv) decreases in chlorideconductance; (v) reduction in synthesis; and the like. A “mutant-CFTRgene” is a gene, or coding sequence, which encodes a mutant-CFTR. Forthe purposes of this application, the terms “genome” and “gene” are usedinterchangeably, e.g. “genome that encodes mutant-CFTR” and “gene thatencodes mutant-CFTR”.

A “gating defective mutant cystic fibrosis transmembrane conductanceregulator protein”, or “gating defective mutant-CFTR” is a mutant-CFTRthat is present on the cell surface and is defective in gating of ionsthrough the channel (e.g., regulation of ion transport). Thus, as usedherein a “gating defective mutant-CFTR” encompasses dysfunctionsassociated with (i) abnormal regulation of conductance; and or (ii)decreases in chloride conductance.

A “mutant-CFTR protein-mediated condition” means any condition, disorderor disease, or symptom of such condition, disorder, or disease, thatresults from or is correlated to the presence of a mutant-CFTR, e.g.,ΔF508-CFTR, e.g., chloride ion impermeability caused by reduced activityof ΔF508-CFTR in ion transport relative to a wild-type CFTR. A“mutant-CFTR protein-mediated condition” encompasses conditions in anaffected subject which are associated with the presence of a ΔF508-CFTRmutation on at least one allele, thus including subjects that carry aΔF508-CFTR mutation on both alleles as well as compound heterozygoussubjects having two different mutant forms of CFTR, e.g., a subject withone copy of ΔF508-CFTR and a copy of different mutant form of CFTR.

Such conditions, disorders, diseases, or symptoms thereof are treatableby specific activation of mutant-CFTR activity, e.g., activation ofmutant-CFTR ion transport. ΔF508-CFTR is correlated to the presence ofcystic fibrosis (CF), and a description of this disease, including itssymptoms, is found in Accession No. 602421 (entitled cystic fibrosistransmembrane conductance regulator; CFTR), and Accession No. 219700(entitled Cystic fibrosis; CF) of the Online Mendelian Inheritance ofMan database, as found at the world wide website of the NationalInstitute of Health at ncbi.nlm.nih.gov. Symptoms of mutant-CFTRprotein-mediated conditions include meconium ileus, liver diseaseincluding biliary tract obstruction and stenosis, pancreaticinsufficiency, pulmonary disease including chronic Pseudomonasaeruginosa infections and other infections of the lung, infertilityassociated with abnormal vas deferens development or abnormal cervicalmucus, and carcinoma including adenocarcinoma. Many subjects that have amutant-CFTR protein-mediated condition are homozygous for a geneencoding a ΔF508-CFTR protein.

A “ΔF508-cystic fibrosis transmembrane conductance regulator protein”,or “ΔF508-CFTR” is the protein that results from the deletion of aphenylalanine residue at amino acid position 508 of the CFTR geneproduct. A “ΔF508-CFTR gene” is a gene, or coding sequence, whichencodes ΔF508-CFTR. A ΔF508-CFTR gene usually results from deletion ofthree nucleotides corresponding to the phenylalanine residue at aminoacid position 508 of the encoded CFTR gene product. For the purposes ofthis application, the terms “genome” and “gene” are usedinterchangeably, e.g. “genome that encodes ΔF508-CFTR” and “gene thatencodes ΔF508-CFTR”. For an example of a gene that encodes ΔF508-CFTR,see, e.g. WO 91/02796.

A “mutant-CFTR activator” as used herein is a compound that increasesthe level of ion transport by a mutant-CFTR relative to ion transport inthe absence of the compound, and particularly with respect to transportof chloride ions. CFTR activators of the invention of particularinterest are those that are specific mutant-CFTR activators, e.g.,compounds that activate mutant-CFTR activity rather than affecting CFTRcellular misprocessing. Mutant-CFTR activators are usually high-affinitymutant-CFTR activators, e.g., have an affinity for mutant-CFTR of atleast about one micromolar, about one to five micromolar, about 200nanomolar to one micromolar, about 50 nanomolar to 200 nanomolar, orbelow 50 nanomolar.

A “gating defective mutant-CFTR activator” as used herein is a compoundthat increases the level of ion transport by a gating defectivemutant-CFTR relative to ion transport in the absence of the compound,and particularly with respect to transport of chloride ions. CFTRactivators of the invention of particular interest are those that arespecific gating defective mutant-CFTR activators, e.g., compounds thatactivate gating defective mutant-CFTR activity rather than affecting,for example, CFTR cellular misprocessing. Gating defective mutant-CFTRactivators are usually high-affinity activators of gating defectivemutant-CFTRs, e.g., have an affinity for a gating defective mutant-CFTR(e.g., ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR) of at leastabout one micromolar, about one to five micromolar, about 200 nanomolarto one micromolar, about 50 nanomolar to 200 nanomolar, or below 50nanomolar.

A “ΔF508-CFTR activator” as used herein is a compound that increases thelevel of ion transport by ΔF508-CFTR relative to ion transport in theabsence of the compound, and particularly with respect to transport ofchloride ions. CFTR activators of the invention of particular interestare those that are specific ΔF508-CFTR activators, e.g., compounds thatactivate ΔF508-CFTR activity rather than affecting CFTR cellularmisprocessing. ΔF508-CFTR activators are usually high-affinityΔF508-CFTR activators, e.g., have an affinity for ΔF508-CFTR of at leastabout one micromolar, about one to five micromolar, about 200 nanomolarto one micromolar, about 50 nanomolar to 200 nanomolar, or below 50nanomolar.

As used herein and in the cystic fibrosis field a “potentiator” refersto a compound that increases a basal level of ion transport by amutant-CFTR (e.g., ΔF508CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR),where the mutant CFTR (in the absence of the compound) exhibitsaberrantly low levels of ion transport relative to wildtype CFTR. Assuch, a “mutant-CFTR potentiator” refers to a potentiator compound that,provides for increased level of ion transport by a mutant-CFTR relativeto ion transport capability of the mutant-CFTR in the absence of thecompounds.

As used herein and in the cystic fibrosis field a “mutant-CFTRcorrector” is a compound that increases the level of ion transport by amutant-CFTR relative to ion transport in the absence of the compound bycorrecting the underlying defect of the CFTR polypeptide, e.g., a defectthat results from post-translational mis-processing (e.g., misfolding).CFTR correctors of the invention of particular interest are those thatfacilitate correction of specific mutant-CFTRs. Mutant-CFTR correctorsare usually exhibit high-affinity for one or more mutant-CFTRs, e.g.,have an affinity for mutant-CFTR of at least about one micromolar, aboutone to five micromolar, about 200 nanomolar to one micromolar, about 50nanomolar to 200 nanomolar, or below 50 nanomolar.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Isolated compounds are usually at leastabout 80%, more usually at least 90% pure, even more preferably at least98% pure, most preferably at least about 99% pure, by weight. Thepresent invention is meant to comprehend diastereomers as well as theirracemic and resolved, enantiomerically pure forms and pharmaceuticallyacceptable salts thereof.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compound(e.g., phenylglycine-containing compound or sulfonamide containingcompound) employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, dileuent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, dileuent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal and the like. In someembodiments the composition is suitable for administration by atransdermal route, using a penetration enhancer other than DMSO. Inother embodiments, the pharmaceutical compositions are suitable foradministration by a route other than transdermal administration.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

A “pharmaceutically acceptable ester” of a compound of the inventionmeans an ester that is pharmaceutically acceptable and that possessesthe desired pharmacological activity of the'parent compound, andincludes, but is not limited to, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl estersof acidic groups, including, but not limited to, carboxylic acids,phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids andboronic acids.

A “pharmaceutically acceptable enol ether” of a compound of theinvention means an enol ether that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable enol ester” of a compound of theinvention means an enol ester that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

“Pro-drugs” means any compound that releases an active parent drugaccording to formula (I) in vivo when such prodrug is administered to amammalian subject. Prodrugs of a compound of formula (I) are prepared bymodifying functional groups present in the compound of formula (I) insuch a way that the modifications may be cleaved in vivo to release theparent compound. Prodrugs include compounds of formula (I) wherein ahydroxy, amino, or sulfhydryl group in compound (1) is bonded to anygroup that may be cleaved in vivo to regenerate the free hydroxyl,amino, or sulfhydryl group, respectively. Examples of prodrugs include,but are not limited to esters (e.g., acetate, formate, and benzoatederivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxyfunctional groups in compounds of formula (I), and the like.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combination thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Halogens of particular interest include chloro and bromogroups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to _(C)4 alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

Overview

The invention provides compounds that increase ion transport in amutant-cystic fibrosis transmembrane conductance regulator protein(mutant-CFTR), e.g., ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, orD1152H-CFTR, and methods of their use in treatment ofmutant-CFTR-mediated diseases and conditions, e.g., cystic fibrosis(CF). Such compounds find use in the study of CFTR ion transport,particularly that of ΔF508-CFTR G551D-CFTR, G1349D-CFTR, andD1152H-CFTR.

In one embodiment, the invention provides high-affinity small-moleculecompounds that increase CF conductance in gating defective mutant-CFTRs,such as ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, and D1152H-CFTR. Thecompounds contemplated by the invention include those of the followingstructural classes: (1) phenylglycine containing compounds; and (2)sulfonamide containing compounds.

The discovery of the subject compounds was based on screening ofnumerous candidate compounds using an assay designed to identifymutant-CFTR activating compounds. A screening of 50,000 diversecompounds identified several compounds and analogs as effectivemutant-CFTR potentiators. The subject compounds are unrelated chemicallyand structurally to previously known mutant-CFTR potentiator compounds.

As such the invention provides compounds that increase ion transportmediated by mutant-CFTR. Without wishing to be bound by this theory, itis speculated, with respect to the ΔF508-CFTR, that the compounds actthrough direct interaction or binding mechanism with ΔF508-CFTR, mostlikely to a site on the first nucleotide binding domain of CFTR wherethe ΔF508 mutation site is located.

The compositions and methods of the invention will now be described inmore detail.

Compositions

Phenylglycine Containing Compounds

The phenylglycine containing compounds describe herein comprise anaromatic- or heteroaromatic nitrogen, a substituted or unsubstitutedphenyl glycine and a substituted or unsubstituted aryl group or acarbonyl group. In specific embodiments, the subject compounds aregenerally described by Formula (I) as follows:

where n R₁ is independently chosen from a substituted or unsubstitutedphenyl group or a substituted or unsubstituted heteroaromatic group; R₂is independently chosen form a hydrogen, a alkyl group, or an ethergroup; R₃ is independently chosen from a hydrogen or an alkyl group, andR₄ is independently chosen from a substituted or unsubstitutedheteroaromatic group; or a pharmaceutically acceptable derivativethereof, as an individual stereoisomer or a mixture thereof. In oneembodiment, R₁ is independently chosen from an unsubstitutedheteroaromatic group or a substituted phenyl group; R₂ is independentlychosen from a hydrogen, a alkyl group, or an ether group; R₃ isindependently chosen from a hydrogen or an alkyl group; and R₄ isindependently chosen form a unsubstituted heteroaromatic group or a or aisopropenylamine group. Exemplary substitutions for R₁, R₂, R₃, and R₄are described in more detail below.

In certain embodiments, the phenylglycine containing compounds aregenerally described by Formula (I), wherein R₁ is a substituted phenylgroup. Such compounds are generally described by Formula (Ia) asfollows:

wherein R₅ is independently chosen from a hydrogen, an alkyl group suchas a substituted or unsubstituted, saturated linear or branchedhydrocarbon group or chain (e.g., C₁ to C₈) including, e.g., methyl,ethyl, isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, or an ether group, such as a methoxyl group or an ethoxylgroup; R₂ is independently chosen from a hydrogen, an alkyl group suchas a substituted or unsubstituted, saturated linear or branchedhydrocarbon group or chain (e.g., C₁ to C₈) including, e.g., methyl,ethyl, isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, or an ether group, such as a methoxyl group or an ethoxylgroup; and R₃ is independently chosen from a substituted orunsubstituted heteroaromatic group, such as an indole group; and R₄ isindependently chosen form a unsubstituted heteroaromatic group or aisopropenylamine group.

In specific embodiments, R₅ is independently chosen from a hydrogen, amethyl group, an isobutanyl group, or a methoxyl group; R₂ isindependently chosen from a hydrogen, a methyl group, or a methoxylgroup; R₃ is independently chosen from a hydrogen or a methyl group; andR₄ is independently chosen from an indole group or a isopropenylaminegroup.

In certain embodiments, the phenylglycine containing compounds aregenerally described by Formula (I), wherein R₁ is a heteroaryl group.Such compounds are generally described by Formula (Ib) as follows:

wherein R₆ is independently chosen from a substituted or unsubstitutedheteroaromatic group, such as a dihydro-benzodioxine group, such as a2,3-dihydro-benzo[1,4]dioxine group; R₂ is independently chosen from ahydrogen, an alkyl group such as a substituted or unsubstituted,saturated linear or branched hydrocarbon group or chain (e.g., C₁ to C₈)including, e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,dodecyl, octadecyl, amyl, 2-ethylhexyl, or an ether group, such as amethoxyl group or an ethoxyl group; R₃ is independently chosen from asubstituted or unsubstituted heteroaromatic group, such as an indolegroup; and R₄ is independently chosen form a unsubstitutedheteroaromatic group or a isopropenylamine group.

In specific embodiments, R₆ is a 2,3-dihydro-benzo[1,4]dioxine group; R₂is independently chosen from a hydrogen, a methyl group, or a methoxylgroup; R₃ is independently chosen from a hydrogen or a methyl group; andR₄ is independently chosen from an indole group or a isopropenylaminegroup.

In some embodiments of the invention, the phenylglycine containingcompounds may comprise a formula of the following:

Sulfonamide Containing Compounds

The sulfonamide containing compounds described herein comprise asubstituted sulfonamide, a substituted heteroaromatic group, and asubstituted formamide. In specific embodiments, the subject compoundsare generally described by Formula (II) as follows:

wherein R₁ is independently chosen form a hydrogen, an alkyl group suchas a substituted or unsubstituted, saturated linear or branchedhydrocarbon group or chain (e.g., C₁ to C₈) including, e.g., methyl,ethyl, isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, or an ether group, such as a methoxyl group or an ethoxylgroup; R₂ is independently chosen from a hydrogen or a substituted orunsubstituted phenyl group; R₃ is independently selected from a an alkylgroup such as a substituted or unsubstituted, saturated linear orbranched hydrocarbon group or chain (e.g., C₁ to C₈) including, e.g.,methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl, dodecyl,octadecyl, amyl, 2-ethylhexyl, an ether group, a substituted orunsubstituted hydrocarbon cyclic ring group, or a substituted orunsubstituted heterocyclic ring; or a pharmaceutically acceptablederivative thereof, as an individual stereoisomer or a mixture thereof.In one embodiment, R₁ is independently chosen from a hydrogen or analkyl group; R₂ is independently chosen form a substituted orunsubstituted phenyl group; R₃ is independently selected from asubstituted or unsubstituted alkyl group, a substituted or unsubstitutedhydrocarbon cyclic ring group, a substituted or unsubstituted(heteroaryl)alkyl group, a substituted or unsubstituted(cycloalkyl)alkyl group, or a substituted or unsubstituted(heterocycloalkyl)alkyl group. Exemplary substitutions for R₁, R₂, andR₃ are described in more detail below.

In specific embodiments, R₁ is independently chosen form a hydrogen; anunsubstituted phenyl group; a mono- or di(halo)phenyl group such as 2-,3-, 4-, or 5-fluorophenyl, 3,4- or 5,6- or 5,7- or 5,8-difluorophenyl; amono- or di-(alkyl)phenyl group, such as a 2-, 3-, 4-, or 5-methylphenylgroup, 2,6- or 3,4- or 5,6- or 5,7- or 5,8-dimethylphenyl; or amono(alkoxy)phenyl group, such as a 2-, 3-, 4-, or 5-methoxyphenyl, 2-,3-, 4-, or 5-ethoxyphenyl, 2-, 3-, 4-, or 5-propoxyphenyl; R₂ isindependently selected from a alkyl group, such as a methyl group, anethyl group, or a propylene group; R₃ is independently selected from aalkyl group, such as a butyl group, a propylene group, an isopentylgroup, and a methoxy-propane; a cycloalkyl group, such as acyclopentane, and a cylcohexane; a (cycloalkyl)alkyl group, such as aethyl-cyclohexene; a (heteroaromatic)alkyl group, such as a3-methyl-furan, and a 2-, 3-, 4-, or 5-methyl-pyridine; or a(heterocycloalkyl)alkyl group, such as a 3-methyl-tetrahydro-furangroup.

In certain embodiments, the sulfonamide containing compounds aregenerally described by Formula (II), wherein the R₁ and R₂ substitutednitrogen is a R₄ group. Such compounds are generally described byFormula (IIa) as follows:

wherein R₄ is a substituted or unsubstituted heterocycloalkyl groupcontaining a nitrogen atom, wherein the heterocycloalkyl group is linkedto the sulfur atom by the nitrogen atom of the heterocycloalkyl group, asubstituted or unsubstituted heterocyclic group; R₃ is independentlyselected from a an alkyl group such as a substituted or unsubstituted,saturated linear or branched hydrocarbon group or chain (e.g., C₁ to C₈)including, e.g., methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl,dodecyl, octadecyl, amyl, 2-ethylhexyl, an ether group, a substituted orunsubstituted hydrocarbon cyclic ring group, or a substituted orunsubstituted heterocyclic ring. Exemplary substitutions for R₄ and R₃are described in more detail below.

In specific embodiments, R₄ is independently select from1,4-Dioxa-8-aza-spiro[4.5]decane group or a 2,3-Dihydro-1H-indole group;and R₃ is independently selected from a alkyl group, such as a butylgroup, a propylene group, an isopentyl group, and a methoxy-propane; acycloalkyl group, such as a cyclopentane, and a cylcohexane; a(cycloalkyl)alkyl group, such as a ethyl-cyclohexene; a(heteroaromatic)alkyl group, such as a 3-methyl-furan, and a 2-, 3-, 4-,or 5-methyl-pyridine; or a (heterocycloalkyl)alkyl group, such as a3-methyl-tetrahydro-furan group.

In some embodiments of the invention, the phenylglycine containingcompounds may comprise a formula of the following:

Analog and Derivative Compounds

Also provided by the invention are analogs and derivatives of thesubject compounds described above. The terms “analog” and “derivative”refers to a molecule which is structurally similar or has the samefunction or activity as the subject phenylglycine containing compoundsor sulfonamide containing compounds of the invention. Such analogs andderivatives of the subject compounds can be screened for efficiency inbinding to and modulating the activity of a mutant CFTR, such asΔF508-CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR.

In some embodiments, in silico modeling can be used to screen3-dimensional libraries of analog or derivative compounds for activityin binding to and modulating the activity of a mutant CFTR, such asΔF508-CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR. An exemplary insilico modeling program suitable for use with the subject method is thePREDICT™ 3D Modeling Technology (Predix Pharmaceuticals, Woburn Mass.),described in greater detail in Becker et al., PNAS 101(31):11304-11309(2004).

Pharmaceutical Preparations Containing Compounds of the Invention

Also provided by the invention are pharmaceutical preparations of thesubject compounds described above. The subject compounds can beincorporated into a variety of formulations for therapeuticadministration by a variety of routes. More particularly, the compoundsof the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers, diluents, excipients and/or adjuvants, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. In mostembodiments, the formulations are free of detectable DMSO (dimethylsulfoxide), which is not a pharmaceutically acceptable carrier, diluent,excipient, or adjuvant, particularly in the context of routes ofadministration other than transdermal routes. Where the formulation isfor transdermal administration, the compounds are preferably formulatedeither without detectable DMSO or with a carrier in addition to DMSO.The formulations may be designed for administration to subjects orpatients in need thereof via a number of different routes, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,intratracheal, etc., administration.

Pharmaceutically acceptable excipients usable with the invention, suchas vehicles, adjuvants, carriers or diluents, are readily available tothe public. Moreover, pharmaceutically acceptable auxiliary substances,such as pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and the like, are readily available to thepublic.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A. R.Gennaro, (2000) Lippincott, Williams & Wilkins. The composition orformulation to be administered will, in any event, contain a quantity ofthe agent adequate to achieve the desired state in the subject beingtreated.

Dosage Forms of Compounds of the Invention

In pharmaceutical dosage forms, the subject compounds of the inventionmay be administered in the form of their pharmaceutically acceptablesalts, or they may also be used alone or in appropriate association, aswell as in combination, with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes,such as intrapulmonary or intranasal delivery.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intrapulmonary intramuscular, intratracheal,intratumoral, subcutaneous, intradermal, topical application,intravenous, rectal, nasal, oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the agent and/or the desired effect. Thecomposition can be administered in a single dose or in multiple doses.

In one embodiment of particular interest, the compounds of the inventionare administered in aerosol formulation via intrapulmonary inhalation.The compounds of the present invention can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Mechanical devices designed for intrapulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.; the “standing cloud” device of InhaleTherapeutic Systems, Inc., San Carlos, Calif.; the AIR inhalermanufactured by Alkennes, Cambridge, Mass.; and the AERx pulmonary drugdelivery system manufactured by Aradigm Corporation, Hayward, Calif. Ofparticular interest are the PARI LC PLUS®, the PARI LC STAR®, and thePARI BABY™ nebulizers by PARI Respiratory Equipment, Inc., Monterey,Calif.

Formulations for use with a metered dose inhaler device may generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid conjugate formulation and mayalso contain a stabilizer such as human serum albumin (HSA). Typically,more than 0.5% (w/w) HSA is added. Additionally, one or more sugars orsugar alcohols may be added to the preparation if necessary. Examplesinclude lactose maltose, mannitol, sorbitol, sorbitose, trehalose,xylitol, and xylose. The amount added to the formulation can range fromabout 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of theconjugate present. Such formulations may then lyophilized and milled tothe desired particle size.

The properly sized particles may then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants may include sorbitantrioleate and soya lecithin. Oleic acid may also be useful as asurfactant. This mixture may then loaded into the delivery device. Anexample of a commercially available metered dose inhaler suitable foruse in the present invention is the Ventolin metered dose inhaler,manufactured by Glaxo Inc., Research Triangle Park, N.C.

Formulations for powder inhalers may comprise a finely divided drypowder containing conjugate and may also include a bulking agent, suchas lactose, sorbitol, sucrose, or mannitol in amounts which facilitatedispersal of the powder from the device, e.g., 50% to 90% by weight ofthe formulation. The particles of the powder may have aerodynamicproperties in the lung corresponding to particles with a density ofabout 1 g/cm.sup.2 having a median diameter less than 10 micrometers,preferably between 0.5 and 5 micrometers, most preferably of between 1.5and 3.5 micrometers. An example of a powder inhaler suitable for use inaccordance with the teachings herein is the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass. The powders for thesedevices may be generated and/or delivered by methods disclosed in U.S.Pat. No. 5,997,848, U.S. Pat. No. 5,993,783, U.S. Pat. No. 5,985,248,U.S. Pat. No. 5,976,574, U.S. Pat. No. 5,922,354, U.S. Pat. No.5,785,049 and U.S. Pat. No. 5,654,007.

For oral preparations, the subject compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

The subject compounds of the invention can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Furthermore, the subject compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Dosages of the Compounds of the Invention

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus, for example, oral dosages may be about ten times the injectiondose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of subject composition to reduce asymptom in a subject animal.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound (s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

Combination Therapy Using the Compounds of the Invention

For use in the subject methods, the subject compounds may be formulatedwith or otherwise administered in combination with otherpharmaceutically active agents, including other CFTR-activating agents.The subject compounds may be used to provide an increase in theeffectiveness of another chemical, such as a pharmaceutical (e.g., otherCFTR-activating agents, or agents that affect cellular misprocessing ofmutant-CFTR), or a decrease in the amount of another chemical, such as apharmaceutical (e.g., other CFTR-activating agents), that is necessaryto produce the desired biological effect.

Examples of other CFTR activating agents include, but are not limitedto, enhancers of intracellular cAMP levels, such as for example, but notlimited to, forskolin, rolipram, 8-bromo-cAMP, theophylline, papaverine,cAMP and salts, analogs, or derivatives thereof. Other examples includebeta agonists, tobramycin (TOBI®, Chiron Inc., Emeryville, Calif.) andcurcumin (Egan et al., (2004) Science 304:600-603).

The compounds described above may also be combined with other therapiesfor CF, including oral corticosteroids, ibuprofen, ribovarin orantibiotics such as dicloxacillin, cephalosporin, cephalexin,erythromycin, amoxicillin-clavulanate, ampicillin, tetracycline,trimethoprim-sulfamethoxazole, chloramphenicol ciprofloxacin,tobramycin, gentamicin, cephalosporins, monobactams and the like.

The compounds described herein for use in combination therapy with thecompounds of the present invention may be administered by the same routeof administration (e.g. intrapulmonary, oral, enteral, etc.) that thecompounds are administered. In the alternative, the compounds for use incombination therapy with the compounds of the present invention may beadministered by a different route of administration that the compoundsare administered.

Kits

Kits with unit doses of the subject compounds, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

Methods

Methods for Increasing Chloride Ion Permeability of a Mutant-CFTR Cell

The invention provides methods for increasing ion permeability of a cellthat produces mutant-CFTR protein, with cells having a gating defectivemutant-CFTR being of interest, with cells having a ΔF508-CFTR,G551D-CFTR, G1349D-CFTR, or D1152H-CFTR being of particular interest. Ingeneral, the method involves contacting the cell with a compound in anamount effective to activate the mutant-CFTR protein and increase ionpermeability of the cell. In one embodiment of particular interest, acompound of the invention is used in the method in combination with asecond mutant-CFTR activator or potentiator.

In many embodiments, the cell mutant-CFTR protein is present on theplasma membrane of the cell. Methods of detecting mutant-CFTR proteinpresence on the plasma membrane are well known in the art and caninclude but are not limited to, for example, labeling a molecule thatbinds to CFTR protein with a fluorescent, chemical or biological tag.Examples of molecules that bind to CFTR protein include, withoutlimitation, antibodies (monoclonal and polyclonal), FAB fragments,humanized antibodies and chimeric antibodies. For an example of anantibody that binds to CFTR protein, see, e.g. U.S. Pat. No. 6,201,107.

In many embodiments, the cell has increased permeability to chlorideions, and the contacting of the cell with a compound of the invention,particularly when provided in combination with a mutant-CFTR activatoror potentiator, increases the rate of chloride ion transport across theplasma membrane of the cell. Contacting the cell with a compound of theinvention usually increases the activity of mutant-CFTR protein toincrease ion transport.

In most embodiments, the ion transport activity of mutant-CFTR, or thepermeability of a cell to ions, is increased by up to about 10%, by upto about 20%, by up to about 50%, by up to about 100%, by up to about150%, by up to about 200%, by up to about 300%, by up to about 400%, byup to about 500%, by up to about 800%, or up to about 1000% or more. Incertain embodiments, where there is no detectable ion transport activityof mutant-CFTR or permeability of a cell to ions, contacting of the cellwith a compound of the invention causes detectable activity ofmutant-CFTR or permeability of a cell to ions.

Activation of mutant-CFTR and/or ion permeability may be measured usingany convenient methods that may use molecular markers, e.g., a halidesensitive GFP or another molecular marker (e.g., Galietta et al., (2001)FEBS Lett. 499, 220-224), patch clamp assays, and short circuit assays.

Suitable cells include those cells that have an endogenous or introducedmutant-CFTR gene. Suitable cells include mammalian cell systems (e.g.,COS, CHO, BHK, 293, 3T3 cells etc.) harboring constructs that have anexpression cassette for expression of mutant-CFTR. The cell used in thesubject methods may be a cell present in vivo, ex vivo, or in vitro. Asused herein, the term “expression cassette” is meant to denote a geneticsequence, e.g. DNA or RNA, that codes for mutant-CFTR protein, e.g.,ΔF508-CFTR. Methods of introducing an expression cassette into a cellare well known in the art, see for example, Sambrook et al., MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY,Vol. 1, 2, 3 (1989).

Methods of Treating Cystic Fibrosis

The invention also provides methods of treating a subject having acondition associated with mutant-CFTR, e.g., cystic fibrosis. Ingeneral, the method involves administering to the subject a compound ofthe invention in an amount effective to activate a mutant-CFTR proteinto increase ion transport and thereby treat the condition. In anembodiment of particular interest, a compound of the invention isadministered in combination with a second mutant-CFTR activator orpotentiator, e.g., a compound that enhances intracellular cAMP, e.g.,forskolin.

The compounds disclosed herein are useful in the treatment of amutant-CFTR-mediated condition, e.g., any condition, disorder ordisease, or symptom of such condition, disorder, or disease, thatresults from the presence and/or activity of mutant-CFTR as compared towild-type CFTR, e.g., activity of mutant-CFTR in ion transport. Suchconditions, disorders, diseases, or symptoms thereof are amenable totreatment by activation of mutant-CFTR activity, e.g., activation ofmutant-CFTR chloride transport. Cystic fibrosis, a hereditary conditionassociated with a mutant-CFTR, e.g., ΔF508-CFTR G551D-CFTR, G1349D-CFTR,or D1152H-CFTR, is an example of a condition that is treatable using thecompounds of the invention. Use of the compounds of the invention incombination with a second mutant CFTR activator or potentiator is ofparticular interest.

Cystic fibrosis is predominantly a disorder of infants, children andyoung adults, in which there is widespread dysfunction of the exocrineglands, characterized by signs of chronic pulmonary disease (due toexcess mucus production in the respiratory tract), pancreaticdeficiency, abnormally high levels of electrolytes in the sweat andoccasionally by biliary cirrhosis. Also associated with the disorder isan ineffective immunologic defense against bacteria in the lungs.

Pathologically, the pancreas shows obstruction of the pancreatic ductsby amorphous eosinophilic concretions, with consequent deficiency ofpancreatic enzymes, resulting in steatorrhoea and azotorrhoea andintestinal malabsorption. The degree of involvement of organs andglandular systems may vary greatly, with consequent variations in theclinical picture.

Nearly all exocrine glands are affected in cystic fibroses in varyingdistribution and degree of severity. Involved glands are of three types:those that become obstructed by viscid or solid eosinophilic material inthe lumen (pancreas, intestinal glands, intrahepatic bile ducts,gallbladder, submaxillary glands); those that are histologicallyabnormal and produce an excess of secretions (tracheobronchial andBrunner's glands); and those that are histologically normal but secreteexcessive sodium and chloride (sweat, parotid, and small salivaryglands). Duodenal secretions are viscid and contain an abnormalmucopolysaccharide. Infertility occurs in 98% of adult men secondary tomaldevelopment of the vas deferens or to other forms of obstructiveazoospermia. In women, fertility is decreased secondary to viscidcervical secretions, but many women with CF have carried pregnancies toterm. However, the incidence of maternal complications increases.

Fifty percent of cystic fibrosis patients with pulmonary manifestationsusually chronic cough and wheezing associated with recurrent or chronicpulmonary infections. Cough is the most troublesome complaint, oftenaccompanied by sputum, gagging, vomiting, and disturbed sleep.Intercostal retractions, use of accessory muscles of respiration, abarrel-chest deformity, digital clubbing, and cyanosis occur withdisease progression. Upper respiratory tract involvement includes nasalpolyposis and chronic or recurrent sinusitis. Adolescents may haveretarded growth, delayed onset of puberty, and a declining tolerance forexercise. Pulmonary complications in adolescents and adults includepneumothorax, hemoptysis, and right heart failure secondary to pulmonaryhypertension.

Pancreatic insufficiency is clinically apparent in 85 to 90% of CFpatients, usually presents early in life, and may be progressive.Manifestations include the frequent passage of bulky, foul-smelling,oily stools; abdominal protuberance; and poor growth pattern withdecreased subcutaneous tissue and muscle mass despite a normal orvoracious appetite. Rectal prolapse occurs in 20% of untreated infantsand toddlers. Clinical manifestations may be related to deficiency offat-soluble vitamins.

Excessive sweating in hot weather or with fever may lead to episodes ofhypotonic dehydration and circulatory failure. In arid climates, infantsmay present with chronic metabolic alkalosis. Salt crystal formation anda salty taste on the skin are highly suggestive of CF.

Insulin-dependent diabetes develops in 10% of adult patients having CF,and multilobular biliary cirrhosis with varices and portal hypertensiondevelops in 4 to 5% of adolescents and adults. Chronic and/or recurrentabdominal pain may be related to intussusception, peptic ulcer disease,periappendiceal abscess, pancreatitis, gastroesophageal reflux,esophagitis, gallbladder disease, or episodes of partial intestinalobstruction secondary to abnormally viscid fecal contents. Inflammatorycomplications may include vasculitis and arthritis.

Any of above symptoms of CF may be treated using the compounds of theinvention, with use of such compounds in combination with a secondmutant-CFTR activator or potentiator being of particular interest.

The above methods may be used to treat CF and its symptoms in humans orin animals. Several animal models for CF are known in the art. Forexample, Engelhardt et al. (J. Clin. Invest. 90: 2598-2607, 1992)developed an animal model of the human airway, using bronchialxenografts engrafted on rat tracheas and implanted into nude mice. Morerecently transgenic models of cystic fibrosis have been produced (e.g.,Clarke et al., Science 257: 1125-1128, 1992; Dorin et al., Nature 359:211-215, 1992). With the recent advances of nuclear transfer and stemcell transformation technologies, the alteration of a wild type CFTRgene in an animal to make it into a mutant-CFTR gene is possible for awide variety of animals.

Many of these animals show human CF symptoms. In particular, many ofthese animals showed measurable defects in ion permeability of airwayand intestinal epithelia, similar to those demonstrable in human CFtissues, and a susceptibility to bacterial infection. Furthermore, mostof the deficient mice had intestinal pathology similar to that ofmeconium ileus. Also, there appeared to be no prenatal loss from littersproduced from crosses between heterozygotes.

Animals suitable for treatment using the subject methods include anyanimal with a mutant-CFTR related condition, particularly a mammal,e.g., non-human primates (e.g., monkey, chimpanzee, gorilla, and thelike), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and thelike), lagomorphs, swine (e.g., pig, miniature pig), equine, canine,feline, and the like. Large animals are of particular interest.Transgenic mammals may also be used, e.g. mammals that have a chimericgene sequence. Methods of making transgenic animals are well known inthe art, see, for example, U.S. Pat. No. 5,614,396. For an example of atransgenic mouse with a CFTR defect, see e.g. WO 94/04669.

Such animals may be tested in order to assay the activity and efficacyof the subject compounds. Improvement in lung function can be assessedby, for example, monitoring prior to and during therapy the subject'sforced vital capacity (FVC), carbon monoxide diffusing capacity(DL_(CO)), and/or room air pO₂>55 mmHg at rest. Significant improvementsin one or more of these parameters are indicative of efficacy. It iswell within the skill of the ordinary healthcare worker (e.g.,clinician) provide adjust dosage regimen and dose amounts to provide foroptimal benefit to the patient according to a variety of factors (e.g.,patient-dependent factors such as the severity of the disease and thelike), the compound administered, and the like).

Subjects Suitable for Treatment

Subjects suitable for treatment with a method of the present inventioninclude individuals having mutant-CFTR protein-mediated conditiondisorder or disease, or symptom of such condition, disorder, or diseasethat results from or is correlated to the presence of a mutant-CFTR,usually two alleles of the mutant CFTR. Moreover, subjects suitable fortreatment with a method of the present invention include individualswith Cystic Fibrosis (CF). Of particular interest in many embodiments isthe treatment of humans with CF.

Symptoms of mutant-CFTR protein-mediated conditions include meconiumileus, liver disease including biliary tract obstruction and stenosis,pancreatic insufficiency, pulmonary disease including chronicPseudomonas aeruginosa infections and other infections of the lung,infertility associated with abnormal vas deferens development orabnormal cervical mucus, and carcinoma including adenocarcinoma.

The compounds of the present invention affect the ion transportcapability of the mutant-CFTR by increasing the reduced level of iontransport mediated by a mutant-CFTR, such as the ΔF508-CFTR, G551D-CFTR,G1349D-CFTR, or D1152H-CFTR. As such, the compounds of the presentinvention have particular clinical utility in treating a subset of CFpatients that have mutations in the CFTR gene that results a mutant-CFTRthat is expressed in the plasma membrane and has reduced chlorideconductance capability or has abnormal regulation of conductance (i.e.,the mutant-CFTR is gating defective). As such, the compounds of thepresent invention have clinical utility in treating CF patients having agating-defective mutant-CFTR, such as ΔF508-CFTR, G551D-CFTR,G1349D-CFTR, or D1152H-CFTR. In addition, the compounds of the presentinvention also have clinical utility in treating CF patients when usedin conjunction with compounds that correct cellular misprocessing of amutant-CFTR, such as ΔF508-CFTR.

CFTR mutations associated with CF are well known in the art. Thesemutations can be classified in five general categories with respect tothe CFTR protein. These classes of CFTR dysfunction include limitationsin CFTR production (e.g., transcription and/or translation) (Class I),aberrant folding and/or trafficking (Class II), abnormal regulation ofconduction (Class III), decreases in chloride conduction (Class IV), andreductions in synthesis (Class V). Due to the lack of functional CFTR,Class I, II, and III mutations are typically associated with a moresevere phenotype in CF (i.e. pancreatic insufficiency) than the Class IVor V mutations, which may have very low levels of functional CFTRexpression. A listing of the different mutations that have beenidentified in the CFTR gene is as found at the world-wide website of theCystic Fibrosis Mutation Database atgenet.sickkids.on.ca/cgi-bin/WebObjects/MUTATION, specificallyincorporated by reference herein in its entirety.

A subject suitable for treatment with a method of the present inventionmay be homozygous for a specific mutant-CFTR, i.e. homozygous subjectswith two copies of a specific mutant-CFTR, e.g., ΔF508-CFTR. Inaddition, subjects suitable for treatment with a method of the presentinvention may also be compound heterozygous for two different CFTRmutants, i.e., wherein the genome of the subjects includes two differentmutant forms of CFTR, e.g., a subject with one copy of ΔF508-CFTR and acopy of different mutant form of CFTR.

In some embodiments of the invention, the mutant-CFTR polypeptide isΔF508-CFTR. In other embodiments of the invention, the mutant-CFTRpolypeptide is G551D-CFTR. In yet other embodiments of the invention,the mutant-CFTR polypeptide is G1349D-CFTR. In still other embodimentsof the invention, the mutant-CFTR polypeptide is D152H-CFTR. Theinvention, however, should not be construed to be limited solely to thetreatment of CF patients having this mutant form of CFTR. Rather, theinvention should be construed to include the treatment of CF patientshaving other mutant forms of CFTR with similar characteristics, thatresult in expression of the mutant-CFTR in the plasma membrane and hasreduced chloride conductance capability or has abnormal regulation ofconductance.

Rational Therapy

The invention also provides rational therapy-based methods for treatinga subject having a condition associated with a mutant-CFTR, e.g., cysticfibrosis. In general, the method involves determining the underlyingCFTR mutation of the patient and selecting a treatment regimen foradministering to the patient based on the CFTR mutation, where thecompound selected for administration is one having activity thatprovides for improved function of the particular CFTR mutant. Ofparticular interest is administration of a compound having enhancedactivity for the particular CFTR mutant of the patient compared to othercompounds of the same genus or class. In this manner, the clinician canmore readily prescribe a successful therapy, based on selection of acompound in light of the CFTR mutation in the patient. Therefore, theselected treatment regimen is more effective and rationally based.Moreover, such rational therapy can significantly reducetherapy-associated toxicity.

As used herein, the process of determining the CFTR mutation of apatient includes any suitable method, of which many are known in theart. Suitable methods include determining the DNA sequence, or bydetecting an RNA transcript corresponding to such DNA sequence, of apolymorphic gene. Various other detection techniques suitable for use inthe present methods will be apparent to those conversant with methods ofdetecting, identifying, and/or distinguishing CFTR mutations. Suchdetection techniques include but are not limited to direct sequencing,use of “molecular beacons” (oligonucleotide probes that fluoresce uponhybridization, useful in real-time fluorescence PCR; see e.g., Marras etal., Genet Anal 14:151 (1999)); electrochemical detection (reduction oroxidation of DNA bases or sugars; see U.S. Pat. No. 5,871,918 to Thorpet al.); rolling circle amplification (see, e.g., Gusev et al., Am JPathol 159:63 (2001)); Third Wave Technologies (Madison Wis.) INVADERnon-PCR based detection method (see, e.g., Lieder, Advance forLaboratory Managers, 70 (2000)).

Accordingly, any suitable detection technique as is known in the art maybe utilized in the present methods to genotype the subject. Furthermore,suitable biological specimens to use for determining the CFTR mutationof the subject are those which comprise cells and DNA and include, butare not limited to blood or blood components, dried blood spots, urine,buccal swabs and saliva.

In practicing the subject methods, once the underlying CFTR mutation ofthe patient is determined, it is used to select a compound that will bemost effective for the underlying CFTR mutation. For example, where thesubject has ΔF508-CFTR mutation, the patient will be administered willbe administered a composition containing a sulfonamide containingcompound in either a mono-drug therapy or in combination with anothercompound as described above. Where the subject has a non ΔF508-CFTRmutation, the patient will be treated with phenylglycine-containingcompound in either a mono-drug therapy or in combination with anothercompound as described above. For example, where the subject has a gatingdefective CFTR mutation, such as a class III mutation (e.g., G551D-CFTR,G1349D-CFTR, or D1152-CFTR), the subject is treated with a phenylglycinecontaining compound in either a mono-drug therapy or in combination withanother compound as described above.

In certain embodiments, once the underlying CFTR mutation of the patientis determined, in silico modeling of the mutant-CFTR performed and 3Dmodels of the subject compounds are screened in order to select acompound having enhanced activity for the particular CFTR mutant of thepatient compared to other compounds of the same genus or class. Anexemplary in silico modeling program suitable for use with the subjectmethod is the PREDICT™ 3D Modeling Technology (Predix Pharmaceuticals,Woburn Mass.), described in greater detail in Becker et al., PNAS101(31):11304-11309 (2004).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

The following methods and materials are used in the examples below.

Cell Lines

Clonal populations of Fischer rat thyroid (FRT) epithelial cells stablyco-expressing human ΔF508-CFTR and the high-sensitivity halide-sensinggreen fluorescent analog YFP-H148Q/I152L (Galietta et al., A.S. (2001)FEBS Lett. 499, 220-224) were generated by liposome transfection andlimiting dilution with Zeocin/G418 selection. More than 100 clones wereevaluated for high fluorescence and ΔF508-CFTR plasma membrane targetingafter growth at 27° C. for 24 hours. For screening, cells were culturedon plastic in Coon's modified F12 medium supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mlstreptomycin, and plated on black 96-well microplates (Corning-Costar3904) at 30,000 cells/well. For short-circuit measurements cells werecultured on Snapwell permeable supports (Corning-Costar) at 500,000cells/insert. Human nasal epithelium cells from CF patients werecultured on Snapwell inserts and allowed to differentiate in ahormone-supplemented medium (Galietta et al., Am. J. Physiol.,275:19723-19728 (1998)). Some measurements were done using stablytransfected FRT cells expressing YFP-H148Q and wildtype- or G551D-CFTR(Galietta et al., (2001) J. Biol. Chem. 276, 19723-19728). Patch clampexperiments were done on ΔF508-CFTR-expressing FRT cells plated in 35-mmPetri dishes.

Compounds

A collection of 50,000 diverse drug-like compounds (purchased fromChemBridge Co.) was used for initial screening. For optimization,compounds identified in the primary screen were purchased from ChemDiv(out of ˜600,000 available compounds). Compounds were prepared as 10 mMstock solutions in DMSO. Secondary plates containing one or fourcompounds per well were prepared for screening (0.25 mM in DMSO).Compounds for secondary analysis were resynthesized, purified, andconfirmed by NMR and liquid chromatography/mass spectrometry.

Screening Procedures

Screening was carried out using a Beckman integrated system containing a3-meter robotic arm, CO₂ incubator containing microplate carousel,plate-washer, liquid handling workstation, bar code reader, deliddingstation, plate sealer, and two FluoStar fluorescence plate readers(Galaxy, BMG Lab Technologies), each equipped with dual syringe pumpsand HQ500/20X (500±10 nm) excitation and HQ535/30M (535±15 nm) emissionfilters (Chroma). Software was written in VBA (Visual Basic forApplications) to compute baseline-subtracted fluorescence slopes (givinghalide influx rates).

For assay of ΔF508-CFTR potentiator activity the incubator (27° C., 90%humidity, 5% CO₂/95% air) was loaded with forty-to-sixty 96-well platescontaining FRT cells. After an 18-24 hour incubation plates were washed3 times with PBS (300 μl/wash) leaving 50 μl PBS. 10 μl of PBScontaining 120 μM forskolin was added, and after 5 min test compounds(0.6 μl of 0.25 mM DMSO solution) were added to each well to give 2.5 μMfinal compound concentrations. After 15 min, 96-well plates weretransferred to a plate reader for fluorescence assay. Each well wasassayed individually for I⁻ influx by recording fluorescencecontinuously (200 ms per point) for 2 seconds (baseline) and then for 12seconds after rapid (<1 s) addition of 160 μL of isosmolar PBS in which137 mM Cl⁻ was replaced by I⁻. I⁻ influx rates were computed frominitial fluorescence versus time-curve slopes (determined by 3^(rd)order polynomial regression) after normalization for total fluorescence(background subtracted initial fluorescence). All compound platescontained negative control (DMSO vehicle alone) and positive controls(genistein, 5 μM and 50 μM). Assay analysis indicated a Z′-factorof >0.7 (Zhang et al., J. Biomol. Screen 4:67-73 (1999)).

Whole-Cell Patch-Clamp

Experiments were performed in the cell-attached configuration of thepatch-clamp technique on FRT cells expressing ΔF508-CFTR. Cells wereseeded at a density of 10⁴ cells/well and grown at 37° C. for 24-48hours and then incubated for 24-48 hours at 27° C. to allow traffickingof the ΔF508 protein to the plasma membrane. Borosilicate glass pipetteswere fire polished to obtain tip resistances of 2-4 ΩM. Currents weresampled at 500 Hz using a patch-clamp amplifier (EPC-7, List, Darmstadt)and low-pass filtered using a 4-pole Bessel filter set at a cutofffrequency of 250 Hz and digitized at 500 Hz using an ITC-16 datatranslation interface (Instrutech). The extracellular (bath) solutioncontained (in mM): 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 mannitol,and 10 TES (pH 7.4). The pipette solution contained (in mM): 120 CsCl, 1MgCl₂, 10 TEA-Cl, 0.5 EGTA, 1 Mg-ATP, and 10 Hepes (pH 7.3). Membraneconductances were monitored by alternating the membrane potentialbetween +80 and −100 mV. Current-voltage relationships were generated byapplying voltage pulses between −100 and +100 mV in 20 mV steps.Analysis of open channel probability (P_(o)), mean channel open time(T_(o)), and mean channel closed time (T_(c)) was done using recordingsof at least three minute intervals (Taddei et al., FEBS Lett. 558:52-56(2004)).

Short-Circuit Current Measurements

Using chamber experiments were performed 7-9 days after platingΔF508-CFTR expressing FRT cells on Snapwell inserts. The basolateralsolution contained (in mM): 130 NaCl, 2.7 KCl, 1.5 KH₂PO₄, 1 CaCl₂, 0.5MgCl₂, 10 glucose, 10 Na-Hepes (pH 7.3). In the apical bathing solution65 mM NaCl was replaced by Na gluconate, and CaCl₂ was increased to 2mM. Solutions were bubbled with air and maintained at 37° C. Thebasolateral membrane was permeabilized with 250 μg/ml amphotericin B.The hemichambers were connected to a DVC-1000 voltage clamp (WorldPrecision Instruments) via Ag/AgCl electrodes and 1 M KCl agar bridgesfor recording short-circuit current.

Synthetic Chemistry

¹H spectra were obtained in CDCl₃ or d₆-DMSO using a Mercury 400 MHzspectrometer. Flash column chromatography was done using EM silica gel(230-400 mesh). Thin layer chromatography was carried out on Merk silicagel 60 F254 plates and visualized under a UV lamp. Microwave reactionswere carried out on an Emrys synthesizer. Representative syntheticschemes for a phenylglycine and sulfonamide follow (FIG. 2, panel B).

For synthesis of compound P-1, to a solution ofN-tert-butoxycarbonyl-N-methylphenylgycine (compound I) (1.26 g, 4.75mmol) at room temperature was added p-isopropylaniline (705 mg, 5.22mmol), 4-(N,N-dimethylamino) pyridine (DMAP) (116 mg, 0.92 mmol) inCH₂Cl₂ (25 mL), and 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide(EDCI, 1.00 g, 5.22 mmol). The reaction mixture was stirred for 2 hoursand then quenched by pouring over saturated NH₄Cl. After extraction withCH₂Cl₂ the organic layer was washed successively with water and brine,dried (Na₂SO₄), and concentrated in vacuo. Column chromatography of thecrude residue gave[(4-isopropylphenylcarbamoyl)-phenylmethyl]-methylcarbamic acidtert-butyl ester (compound IIA) as a white solid (1.67 g, 92%). CompoundIIA (300 mg, 0.785 mmol) was dissolved in a minimal quantity oftrifluoroacetic acid (TFA), maintained at room temperature for 15 min,poured over aqueous NaHCO₃, and extracted with CH₂Cl₂. Washing, dryingand evaporation of the organic layer gave compound II as a yellow oil(218 mg, 98%). To a mixture of compound II (177 mg, 0.620 mmol),indole-3-acetic acid (114 mg, 0.651 mmol) and DMAP (15 mg, 0.124 mmol)in CH₂Cl₂ (5 mL), EDCI (131 mg, 0.682 mmol) was added at roomtemperature. The reaction mixture was worked up as for compound IIA andrecrystallized from CH₂Cl₂: MeOH (9:1) to give compound P-1 as a whitesolid (1.67 g, 92%). Mass (ES+): M/Z=440 [M+1]⁺; ¹H NMR δ 1.21 (d, ⁶H,J=6.9 Hz), 2.85 (sep, ¹H, J=6.9 Hz), 2.95 (s, ³H), 3.91 (s, ²H), 6.55(s, ¹H), 7.08-7.40 (m, ¹³H), 7.59 (d, ¹H, J=7.8 Hz), 7.88 (bs, ¹H), 8.13(bs, ¹H).

For synthesis of compound S-3, compound III (Blus, Dyes and Pigments41:149-157 (1999)) (2.21 g, 8.0 mmol) and diethylethoxymethylenemalonate(1.81 g, 8.4 mmol) were dissolved in tetrahydrofuran (THF) (4 mL), andthe solution was heated to 140° C. for 30 min until the THF and ethanolby-product evaporated. The residue was diluted with ethyl acetate(EtOAc), washed with brine, dried with Na₂SO₄, and evaporated todryness. Flash chromatography gave light yellow solid compound IIIB(3.29 g, 90%). To a solution of phenyl ether (Ph₂O, 3 mL) and compoundIIIB (130 mg, 0.30 mmol) in an Emrys microwave reaction vessel was added4-chlorobenzoic acid (1 mg, 0.02 mmol). The solution was microwaveirradiated at 250° C. for 75 min. The white precipitate was filtered andwashed with hexane to yield compound IV (48 mg, 42%). To an Emrysmicrowave reaction vessel (0.2-0.5 mL) containing compound IV (65 mg,0.083 mmol) was added o-methoxybenzyl amine (200 mg, 1.4 mmol) andmicrowave irradiated at 180° C. for 30 min. The resulting solution wasdiluted with dichloromethane and water, and extracted with EtOAc threetimes. After washing, drying and evaporation, the residue was purifiedby flash chromatography giving compound S-3 as a white powder (27 mg,35%). Mass (ES+): M/Z=492 [M+1]⁺; ¹H NMR CDCl₃ δ 1.08 (t, ³H, J=7.2 Hz),3.65 (q, ²H, J=7.2 Hz), 3.79 (s, ³H), 4.70 (d, ²H, J=6.0 Hz), 6.81 (m,²H), 7.02 (m, ²H), 7.16 (td, ¹H, J=8.0, 1.6 Hz), 7.23 (d, J=7.2 Hz),7.29 (m, ²H), 7.37 (d, ¹H, J=8.4 Hz), 7.53 (dd, ¹H, J=8.8, 2.0 Hz), 8.77(d, ¹H, J=2.0 Hz), 8.83 (d, J=6.4 Hz), 10.74 (t, ¹H, J=5.6 Hz), 12.30(d, ¹H, J=4.4 Hz).

Assay of cAMP

cAMP activity was measured using the BIOTRAK enzymatic immunoassay(Amersham) of FRT cell lysates after incubation with the compounds for10 minutes in the presence of 0.5 μM forskolin.

Pharmacokinetics

To increase compound solubility, potentiators were dissolved in aliposomal formulation containing 5 mg potentiator in 21.3 mghydrogenated soy phosphatidylcholine, 5.2 mg cholesterol, 8.4 mgdistearoylphosphatidylglycerol, and 90 mg sucrose in 5 ml PBS. A bolusof potentiator-containing solution (5 mg/kg) was administeredintravenously in rats over 1 min (male Sprague-Dawley rats, 360-420grams) by a jugular vein catheter. Arterial blood samples (˜1 ml) wereobtained at predetermined times for LCMS analysis.

Liquid Chromatography/Mass Spectrometry (LCMS)

For analysis of blood samples, collected plasma was chilled on ice, andice-cold acetonitrile (2:1 v:v) was added to precipitate proteins.Samples were centrifuged at 4° C. at 20,000 g for 10 min. Supernatants(supplemented with sulforhodamine 101 as internal standard) wereanalyzed for compound P-1 and compound S-3 by extraction with C-18reversed-phase cartridges (1 ml, Alltech Associates, Inc. Deerfield,Ill.) by standard procedures. The eluate was evaporated, and the residuewas reconstituted in 100 p.1 of mobile phase for HPLC analysis.Reversed-phase HPLC separations were carried out using a Supelco C18column (2.1×100 mm, 3 μm particle size) connected to a solvent deliverysystem (Waters model 2690, Milford, Mass.). The solvent system consistedof a linear gradient from 20% CH₃CN/10 mM KH₂PO₄, pH 3 to 95% CH₃CN/10mM KH₂PO₄, pH 3 over 10 min, followed by 6 min at 95% CH₃CN/20 mM NH₄OAc(0.2 ml/min flow rate). Compounds P-1 and S-3 were detected at 256 nm,after establishing a linear standard calibration curve in the range of20-5000 nM. The detection limit was 10 nM and recovery was >90%. Massspectra were acquired on a mass spectrometer (Alliance HT 2790+ZQ) usingnegative ion detection, scanning from 200 to 800 Da (Sonawane et al., J.Pharm. Sci. 94:134-143 (2004)).

Stability in Hepatic Microsomes

Compounds P-1 and S-3 (10 μM each) were incubated separately with aphosphate buffered (100 mM) solution of rat liver microsomes (2 mgprotein/ml, Sigma) containing NADPH (0 or 1 mM) for 60 min at 37° C.After 60 min the mixture was chilled on ice, and 0.5 ml of ice-coldacetonitrile was added to precipitate the proteins for LCMS analysis asdescribed above.

Example 1 Screening Assays and Structure-Activity Relationship

The high-throughput screen was designed to identify compounds thatactivated ΔF508-CFTR when expressed at the cell plasma membrane. FRTepithelial cells co-expressing ΔF508-CFTR and a high sensitivity yellowfluorescent protein-based halide indicator were incubated at 27° C. for24 h to permit ΔF508-CFTR plasma membrane targeting (FIG. 1, panel A).After washing, forskolin (20 μM) and test compounds (2.5 μM) were addedto individual wells of 96-well plates. The influx assay was carried out˜15 min later by measurement of the time course of decreasing YFPfluorescence after creation of an inwardly-directed I⁻ gradient. A highconcentration of forskolin was used to identify ΔF508-CFTR potentiatorsthat may interact directly with ΔF508-CFTR rather than alter cAMPconcentration. Since activation of CFTR requires cAMP stimulation,forskolin, an enhancer of cAMP, was added to the in vitro models inorder to mimic the cellular cAMP stimulation. Each plate also containedpositive control wells in which a dose-response was done for genistein,a known (though low potency) ΔF508-CFTR potentiator. The screeningrevealed many compounds that at 2.5 μM increased I⁻ influx as much asthe reference compound genistein at 50 μM, and substantially greaterthan forskolin (20 μM) alone (see FIG. 1, panel B). FIG. 2, panel A,depicts representative structures of the two classes of compoundsidentified by the subject screen.

The strong potentiators were subjected to secondary analysis to select asubset for further analysis. More than 300 structural analogs wereevaluated to establish structure-activity relationships and to identifycompounds with improved potency. Dose-response studies were done todetermine K_(a) and V_(max), with representative data shown in FIG. 3,panel A (phenylglycine containing compounds) and panel B (sulfonamidecontaining compounds). Dose response data from the fluorescence assayfor the most active compounds of each class is shown in FIG. 3, panel C,with data for comparison shown for genistein and thetetrahydrobenzothiophene ΔF508_(act)-02. Many compounds were identifiedthat activated ΔF508-CFTR chloride conductance by 50% at concentrationsunder 1 Several of these compounds are shown in Tables 1, along withdata as to the activity of these compounds as ΔF508-CFTR potentiators.By short-circuit current analysis, the most potent compounds activatedΔF508-CFTR chloride strongly at concentrations well under 100 nM. Themaximal current was similar to that of tetrandrobenzothiophene andflavone-type compounds.

The results of the structure-activity relationship are summarized Table1 and Table 2, and the principle conclusions of the structure-activityrelationship are provided in FIG. 2, panel C. Active phenylglycinecontaining compounds contained a disubstituted glycyl amine with amideof aromatic amines. Substitutions at R₁ had relatively little effect oncompound activity. Most active compounds had as R₁ 4-isopropylphenyl,with reduced activity for R₁ as benzo[3,4-b][1,4]dioxane in (P-2, P-4)or 4-methoxyphenyl (P-5). Evaluation of R₂ substitutions indicated thatreplacement of hydrogen by methyl (PG-07) or methoxy (PG10) stronglyreduced potency. The R2 phenyl group appeared to be important foractivity as its replacement by indol-3-methyl reduced activity. Allpotent compounds had as R3 a methyl, as its replacement by hydrogen(PG-06) or furfuryl-2-methyl reduced activity. Most active compounds hadas R4 an indolyl-3-acetyl, as substitution by thiophene-2-acetyl ordiphenyl acetyl resulted in loss of activity. Thus, greatest ΔF508-CFTRactivating potency was produced by hydrophobic R1, R2, and R3, with R4as indolyl-2 (or 3)-acetyl.

The results of the structure-activity relationship analysis ofsulfonamides show that the requirement of 3-carboxamide and 6-aminosulfogroups. All quinolone compounds had as R₁ hydrophobic groups such asalkoxy, dialkyl, alkyl, and halo substituted phenyl or cyclohexyl groups(S-1). Greatest activity was found for R₂ as non-polar alkyl chains(ethyl, methyl, 2-propenyl). The most potent compounds (S-2, S-3, andS-4) contained an ethyl group at R₂ in combination with phenyl as R₁,and linear alkyl group as R₃. Substitutions at R₃ with non-polar linearor branched alkyl or cycloalkyl groups improved activity. In general,greatest potency was found with hydrophobic-nonpolar substitutions onsulfonamide and carboxamide moieties

TABLE 1 Structure-activity relationship analysis of phenylglycinecontaining compounds

Compd R1 R2 R3 R4 Ka (μM) P-1 4-Isopropyl-Ph H Me Indol-3-actyl 0.30 P-22,3-diH-1,4-benzodioxin-6-yl H Me Ac—NHCH₂CO— 0.30 P-3 4-Isopropyl-Ph4-OMe Me Indol-3-actyl 0.34 P-4 2,3-diH-1,4-benzodioxin-6-yl H MeIndol-3-acetyl 0.40 P-5 4-OMe—Ph H Me Indol-3-acetyl 0.70 P-64-Isopropyl-Ph H H Indol-3-acetyl 0.88 P-7 1,3-benzodioxol-5-yl 4-Me MeIndol-3-acetyl 1.33 P-8 4-OMe—Ph 4-OMe Me Indol-3-acetyl 2.13 P-92,3-diH-1,4-benzodioxin-6-yl 4-Me H Indol-2-acetyl 2.33 P-102,3-diH-1,4-benzodioxin-6-yl 4-OMe Me Indol-3-acetyl 2.71 P-114-Isopropyl-Ph 4-Me 2-Furanylmethyl Indol-3-acetyl Moderate P-124-OMe—Ph 4-Me Me Indol-3-acetyl Activity P-13 4-OMe—Ph 4-Me2-Furanylmethyl Indol-3-acetyl P-14 4-OMe—Ph 4-OMe 2-FuranylmethylIndol-3-acetyl P-15 3-Me—Ph Indol-3-CH₂—* H 2,2-Di-Ph-acetyl P-163,4-Di-Me—Ph Indol-3-CH₂—* H 2,2-Di-Ph-acetyl *—Ph—R2 group is replacedby indol-3-CH₂— group

TABLE 2 Structure-activity relationship analysis of sulfonamidecontaining compounds

Compd R1 R2 R3 Ka uM) S-1 2-OEt—Ph Me 2-propenyl 0.30 S-2 Ph EtCycloheptyl 0.02 S-3 Ph Et 2-OMe—Ph—CH₂ 0.03 S-4 Ph Et Cyclohexyl 0.03S-5 OEt—Ph Me n-Pentyl 0.06 S-6 Ph 2-propenyl n-butyl 0.11 S-7 Ph2-propenyl Cycloheptyl 0.12 S-8 2,5-Di-Me—Ph Me 2-Pyridinylmethyl 0.13S-9 Ph Et (3-OMe)-propyl 0.14 S-10 —CH₂—CH₂—CH(Me)—CH₂—CH₂— H3[(N-(n-butyl)phenylamino)propyl 0.14 S-11 Ph 2-propenyl2-Pyridinylmethyl 0.16 S-12 Ph 2-Propenyl n-Hexyl 0.19 S-13 2-Me—Ph Men-butyl 0.20 S-14 2-EtO—Ph Me (Tetrahydro-2-furanyl)methyl 0.20 S-153-Me—Ph Me n-pentyl 0.22 S-16 Ph Et 2-(1-cyclohexen-1-yl)ethyl 0.24 S-17Ph Et (Tetrahydro-2-furanyl)methyl 0.24 S-18 2-Et—Ph Me2-Pyridinylmethyl 0.27 S-19 2,5-Di-Me—Ph Me 3-OMe-propyl 0.29 S-202,6-Di-Me—Ph Me n-Butyl 0.33 S-21 4-F—Ph Et Cyclopentyl 0.33 S-224-Et—Ph Me 2-(Di-OEt)ethyl 0.36 S-23 2-OMe-5-Cl—Ph Me2(1-Cyclohexene-1-yl)ethyl 0.37 S-24 Et Et 1,3-Benzodioxol-5-lymethyl0.38 S-25 3-Me—Ph Me 1-Me-propyl 0.44 S-26 2-Et—Ph Me 1-Me-Propyl 0.44S-27 Ph Et 2-Furanylmethyl 0.46 S-28 3-Me—Ph Et 3-OMe-Propyl 0.48 S-293-Me—Ph Me 2(1-cyclohexene-1-yl)ethyl 0.49 S-30 4-F—Ph Et(Tetrahydro-2-furanyl)methyl 0.54 S-31 3-Me—Ph Me n-Propyl 0.56 S-32—(2-Benzo-CH₂—CH₂)— ** H Cyclohexyl 0.57 S-33 Ph Et 4-Me—Ph—CH₂— 0.59S-34 Cyclohexyl Me (Diethoxycarbonyl)methyl 0.59 S-35 3-Me—Ph Et2-OMe—Ph—CH₂— 0.60 S-36 2-Et—Ph Me 3-OEt-propyl 0.62 S-37 Ph 2-Propenyl2-Furanylmethyl 0.65 S-38 4-Cl-2-F—Ph Me (Tetrahydro-2-furanyl)methyl0.66 S-39 Et Et 4-OMe—Ph—CH₂— 0.66 S-40 3-Me—Ph Et 3-Me-n-Butyl 0.72S-41 Et Et n-Butyl 0.74 S-42 —(2-Benzo-CH₂—CH₂)— ** H 3-Me-butyl 0.76S-43 2-Et—Ph Me (2-OMe)-ethyl 0.77 S-44 —CH₂—CH₂—C(OCH₂—CH₂—O)CH₂—CH₂— H(2-OMe—Ph)methyl 0.80 S-45 4-Br—Ph Me (1-Me)propyl 0.81 S-463,4-Di-Me—Ph Me Propyl 0.84 S-47 2-Me—Ph Me 3-Me-Butyl 0.87 S-48—CH₂—CH₂—C(OCH₂—CH₂—O)CH₂—CH₂— H n-Pentyl 0.88 S-49—CH₂—CH₂—CH(Me)—CH₂—CH₂— H n-Pentyl 0.88 S-50 4-F—Ph Et 3-OMe-Propyl1.02 S-51 3-Me—Ph Et (Tetrahydro-2-furanyl)methyl 1.11 S-52 2-Et—Ph Me2-Propenyl 1.14 S-53 Ph Et Isopropyl 1.16 S-54 2-OEt—Ph Me n-Octanyl1.16 S-55 4-F—Ph Me Propyl 1.25 S-56 —CH₂—CH(Me)—CH₂—CH₂—CH₂— H n-Butyl1.27 S-57 Ph Et n-Hexyl 1.28 S-58 2-Et—Ph Me 2-(Di-OEt)ethyl 1.28 S-592-Me—Ph Me 1-Me-Propyl 1.28 S-60 2-F-4-Cl—Ph Me (3-OEt)-n-Propyl 1.37S-61 2,6-Di-Me—Ph Me (3-OMe)-n-Propyl 1.42 S-62 2-F-4-Cl—Ph Me n-Propyl1.45 S-63 —CH₂—CH₂—CH(Me)—CH₂—CH₂— H n-Hexyl 1.53 S-64 4-F—Ph Et n-Butyl1.56 S-65 2-Me—Ph Me 3-OEt-Propyl 1.66

Example 2 Short-Circuit Current Analysis

Short-circuit current analysis was done on each of these compounds toconfirm bona fide activation of ΔF508-CFTR Cl⁺ currents. Experimentswere done after basolateral membrane permeabilization and in thepresence of a transepithelial Cl⁺ gradient, so that short-circuitcurrent represents apical membrane current. Representative data areshown in FIG. 4, panel A. CFTR-mediated chloride currents measured inFRT cells expressing ΔF508-CFTR. Cells were plated on a permeablesupport to generate a polarized epithelium, cultured for 5-7 days, andthen incubated at 27° C. for 24 hours. Transepithelial chloride currentwas measured in a modified Ussing chamber in the presence of a chloridegradient. Cells were maximally stimulated with forskolin (20 μM) andthen with the indicated concentrations of the phenylglycine containingcompound P-1 and the sulfonamide containing compound S-1. Specificactivation of CFTR is demonstrated by the block of current caused by thethiazolidinone CFTR inhibitor CFTR_(inh)-172. The results show that thephenylglycine containing compound P-1 and the sulfonamide containingcompound S-1 gave ΔF508-CFTR currents with potencies better than 100 nM,and maximal currents comparable to or greater than that produced by 50μM genistein (see FIG. 3, panel B).

An interesting observation was that these new potentiators increased thesensitivity of ΔF508-CFTR to forskolin at low concentrations. FIG. 5depicts the results with phenylglycine containing compounds andsulfonamide containing compounds showing potentiation of the response ofΔF508-CFTR to forskolin. FIG. 5, Panel A shows the representative tracesobtained from Ussing chamber experiments show the effect of forskolin atincreasing concentrations in the presence and the absence of thephenyglyicine containing compound P-1 (100 nM). FIG. 5, panel A showsthat forskolin alone produces a small increase in current, with littleeffect at 2 μM and a larger effect at 20 μM (top). However, afterpreincubation with the phenylglycine potentiator, low concentrations offorskolin (0.5 μM) produce substantial currents (bottom). FIG. 5, PanelB shows a summary of similar experiments for the phenylglycinecontaining compound P-1 and the sulfonamide containing compound S-1showing significant increase in current induced by low concentrations offorskolin.

Example 3 cAMP Analysis

An analysis of compound specificity was also performed. Cells wereincubated with potentiators in the presence of a low concentration offorskolin (0.5 μM), lysed, and assayed for cAMP. The results show thatthe compounds P-1 and S-1 did not increase cAMP above the level inducedby forskolin 0.5 μM alone (FIG. 6, panel A), whereas the compoundCFTR_(act)-16, an indirect activator of CFTR (Ma et al., J. Biol. Chem.277:37235-37241 (2002)), strongly increased cAMP. In addition; multipledrug resistance protein-1 (MDR-1) activity was assayed by intracellularaccumulation of the fluorescent probe rhodamine 123. The wo cell linesused in the assay were the parental human tracheal cell line 9HTEo-, andits multidrug resistant subclone 9HTEo-/Dx that strongly expresses MDR-1(Rasola et al., J. Biol. Chem. 269:1432-1436 (1994)). The results showthat the 9HTEo-/Dx cells accumulate much less rhodamine 123 than 9HTEo-cells as a consequence of MDR-1 mediated dye extrusion. Dye accumulationwas increased significantly by the MDR-1 inhibitor verapamil, but wasnot affected by compounds P-1 or S-1 (FIG. 6, panel B). In addition,effects on the UTP/calcium activated Cl⁻ channel were measured fromshort circuit current measurements on human bronchial epithelial cells.The results show that compounds P-1 or S-1 had no effect on themagnitude or kinetics of the calcium-activated Cl⁻ current (FIG. 6,panel C).

Based on the measurements of cellular cAMP concentrations, the resultsshow that the apparent synergy of the compounds with forskolin is notdue to cAMP elevation. The results show a direct interaction between thephenylglycine containing compounds and the sulfonamide containingcompounds with ΔF508-CFTR. The lack of effect of the compounds in theabsence of cAMP elevating agents and the apparent synergy with cAMPelevating agents are favorable properties in that near-relative CFTRregulation is recapitulated.

Example 4 Patch-Clamp Analysis

Patch-clamp analysis was done to establish the electrophysiologicalmechanism of ΔF508-CFTR activation. Representative single channelrecordings shown in FIG. 7, panel A indicate strong activation ofΔF508-CFTR chloride channels at 100 nM concentrations of thephenylglycine and sulfonamide potentiators. Channel open probably (Po)was increased without change in channel unitary conductance. The subjectcompounds increased Po greatly over that by forskolin alone, to levels(˜0.4) measured for wild-type CFTR measured under the same conditions.

FIG. 7, panel A shows the results of the patch-clamp analysis. A.Cell-attached patch-clamp recordings show ΔF508-CFTR channel activity inthe presence of forskolin (20 μM) (top) and after addition of thephenylglycine containing compound P-1 or sulfonamide containing compoundS-1 (100 nM, bottom). The closed channel level is indicated by a dashedline. Downward deflections indicate channel opening. The large increasein channel activity caused by the potentiators seen by the appearance ofmultiple channel openings of long duration. FIG. 7, panel B shows theaveraged channel open probabilities (P_(O)) (SEM) from data as in FIG.7, panel A. In addition, analysis of gating kinetics shows that theincrease in Po was due to a reduction in mean channel closed time(T_(C)) rather than an increase in mean channel open time (T_(O)) (FIG.7, panel B).

Example 5 Native Human Airway Epithelial Cells

To demonstrate that the compounds identified by screening humanΔF508-CFTR in transfected epithelial cells also were effective in nativehuman airway cells, short-circuit current measurements were done onprimary cultures of nasal epithelial cells from a ΔF508 homozygoussubject. Representative short-circuit data are shown in FIG. 8. MaximalΔF508-CFTR activation was found for potentiator concentrations less than500 nM, showing that the potentiators are effective in native humancells.

Human nasal epithelial cells from ΔF508 homozygote subjects werecultured as polarized monolayers on permeable supports fortransepithelial short-circuit current measurement. After blocking theepithelial Na⁺ channel with amiloride, forskolin (20 μM) was applied,followed by genistein, compound P-1, or compound S-1. CFTR_(inh)-172 wasapplied at the end of each study to determine total CFTR-dependentcurrent. Cells maintained at 37° C. had little CFTR current, inagreement with the expected intracellular retention of ΔF508-CFTR. Lowtemperature rescue by incubation at 27° C. for 20-24 hours producedgreater ΔF508-CFTR current, with significant activation by compounds P-1and S-1 at nanomolar concentrations (FIG. 8, panel A). Stimulation byforskolin plus compound P-1 or compound S-1 was blocked byCFTR_(inh)-172. Genistein was comparably effective but at much higherconcentrations.

In addition, primary cell cultures from subjects carrying CFTR mutationscausing pure gating defects were also tested. For these studies cellswere cultured at 37° C. The results show that nasal epithelial cellsfrom a subject with the G551D mutation (Zegarra-Moran et al., Br. J.Pharmacol. 137:504-512 (2002)) had a large response to compound P-1after forskolin stimulation (FIG. 8, panel B). Cells from a subjecthaving D1152H and ΔF508 CFTR mutations were also tested. The D1152Hmutation affects the second nucleotide binding domain and causes adecrease in channel activity (Vankeerberghen et al., FEBS Lett. 437:1-4(1998)). The results show that the D1152H/ΔF508 cells maintained at 37°C. cells had large CFTR currents in response to compound P-1 (FIG. 8,panel C).

Example 6 Correction of Defective Gating

To demonstrate that the phenylglycine containing compounds andsulfonamide containing compounds are also effective in activating otherforms of mutant CFTR, the compounds were tested with the “class III”gating defective mutant CFTRs G551D-CFTR and G1349D-CFTR. The G551D-CFTRand G1349D-CFTR mutations produce a severe gating defect withoutimpairment in protein trafficking (Gregory et al., MCB 11:3886-3893(1991). These mutations affect the glycine residues in NBD1 and NBD2that are highly conserved in ATP-binding cassette proteins (Hyde et al.,1990; Logan et al., 1994). The G551D-CFTR gating defective mutant is themost common CFTR gating mutant that causes CF.

Experiments were done after basolateral membrane permeabilization and inthe presence of a transepithelial Cl⁺ gradient, so that short-circuitcurrent represents apical membrane Cl⁺ current. Representative data areshown in FIG. 9, panel A. CFTR-mediated chloride currents measured inFRT cells expressing either G551D-CFTR (FIG. 9, panel A, left panel) orG1349D-CFTR (FIG. 9, panel A, right panel). Cells were plated on apermeable support to generate a polarized epithelium, cultured for 5-7days, and then incubated at 27° C. for 24 hours. Transepithelialchloride current was measured in a modified Ussing chamber in thepresence of a chloride gradient. Cells were maximally stimulatedforskolin and then with the indicated concentrations of thephenylglycine containing compound P-1 (bottom portion of each panel) orgenestein, a flavone compound known at high concentrations to correctgating defective mutant CFTRs (top portion of each panel). Specificactivation of CFTR is demonstrated by the block of current caused by thethiazolidinone CFTR inhibitor CFTR_(inh)-172.

The G551D and G1349D mutant CFTRs produced little Cl⁻ current afteraddition of maximal forskolin (FIG. 9, panels A and B). Genistein, aknown activator of G551D- and G1349D-CFTR, increased Cl currentsubstantially, albeit at high micromolar concentrations (FIG. 9, panelsA and B, top panels). Compound P-1 produced large currents in bothG551D- and G1349D-CFTR expressing cells as shown in FIG. 9, panels A andB (bottom panels), and summarized in FIG. 9, panels C and D. Thecurrents were sensitive to CFTR_(inh)-172 and not seen innon-transfected cells. The results show that the activating potency ofP-1 was found to be 50-100 times better than that of genistein.

The results show that the phenylglycine containing compounds correcteddefective gating in a number of CF-causing CFTR mutants including ΔF508,G551D, G1349D and D1152H. The G551D and G1349D mutations affect criticalglycine residues in nucleotide binding domains 1 and 2 of CFTR,respectively (Hyde et al., Nature 346:362-365 (1990)), producing a puregating defect of greater severity than that in ΔF508-CFTR (Gregory et.al., MCB 11:3886-3893 (1991); Logan et. al., J. Clin. Invest. 94:228-236(1994); Zegarra-Moran et. al., Br. J. Pharmacol. 137:504-512 (2002);Derand et. al., JBC 277:35999-36004 (2002)). Forskolin alone producedlittle activation of these mutant CFTRs even at high concentrations,whereas compound P-1 after application of forskolin produced a >10-foldelevation in current. The results show that the K_(d) for compound P-1for G551D-CFTR activation was ˜1 μM, approximately 100-fold better thanthat of genistein. The potency for activation of G1349D-CFTR by compoundP-1 was even better, ˜40 nM. In contrast to the ΔF508 mutation, othercystic fibrosis mutations, which number >1000, have a relatively verylow frequency. The fraction of CF mutations that cause a pure gatingdefect (class III mutants) is unknown but is likely to be substantial.The results show that the phenylglycine containing compounds can be usedin mono-drug therapy for many of these mutations.

Example 7 Correction of Defective Gating In Nasal Polyp Epithelial Cells

To demonstrate that the phenylglycine containing compounds identified byscreening human ΔF508-CFTR in transfected epithelial cells also wereeffective in correcting defective gating native human tissues,short-circuit current measurements were done on cultures of nasal polypepithelial cells from a CF patient with the G551D-CFTR mutation.Representative short-circuit data are shown in FIG. 10. MaximalG551D-CFTR activation was found for potentiator concentrations less than10 μM, indicating that the potentiators are effective in human nasalpolyp epithelial cells.

FIG. 10 shows the results of the G551D-CFTR activity in nasal polypepithelial cells from G551D-CFTR human subject in response to thesubject compounds. Epithelial cells were plated on permeable supports togenerate polarized monolayers resembling the epithelium in vivo. Afterblocking the epithelial sodium channel with amiloride, CFTR-dependentchloride secretion was stimulated with forskolin at maximalconcentration. The phenylglycine containing compound P-1 furtherincreased CFTR-mediated currents. This effect was fully blocked by CFTRinhibitor CFTR_(inh)-172.

Example 8 Hepatic Clearance of Compounds

To predict hepatic clearance of compounds P-1 and S-3, in vitroincubations were done with rat hepatic microsomes for 1 hour at 37° C.in the absence (control) and presence of NADPH, followed by LCMSanalysis. Compound S-3 was chosen for these studies as the most potentof the sulfonamide containing compounds. FIG. 11, panel A (top, left andright), shows representative HPLC chromatograms, with compound P-1eluting at 7.85 min, and its two major metabolites (M1 and M2) elutingat 6.88 and 7.16 min. Mass spectrometry identified the originalcompound, and M1 and M2 with m/z 456 (˜PG-01+OH; [M+1]⁺) and 472(˜P-1+2OH; [M+1]⁺), respectively (FIG. 11, panel A, top, middle). Aminor metabolite was also detected at 7.43 min with m/z 428.Approximately 90% of compound P-1 was metabolized after incubation withmicrosomes for 1 hour in the presence of NADPH, and non-metabolizedcompound P-1 was not detectable after 2 hours. FIG. 11, panel A (bottom,left and right), shows the HPLC profile for compound S-3 and its twomajor metabolites eluting at 7.44 min and 7.16/6.77 min, respectively,with corresponding molecular ion peaks (FIG. 11, panel A, bottom,middle) at m/z 492 (S-3, [M+1]⁺), 508 (˜S-3+0H, [M+1]⁺) and 389.Compound S-3 was ˜35% degraded after a 1 hour incubation with livermicrosomes in presence of NADPH.

Example 9 Pharmacokinetic Analysis of Compounds

Pharmacokinetic analysis of P-1 and S-3 in rats was done by serialmeasurements of plasma concentrations after single bolus infusions (5mg/Kg). FIG. 11, panel B (left), shows HPLC chromatograms for compoundsP-1 and S-3 (each at 50 nM added to control plasma and supplemented withsulforhodamine 101 as internal standard), demonstrating the sensitivityof the assay. Compound P-1 pharmacokinetics fitted a two-compartmentmodel with half-times of approximately 0.2 hour and 1 hour, whereascompound S-3 clearance had elimination half-time of approximately 1.3hours (FIG. 11, panel B, right).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-23. (canceled)
 24. A method of treating a subject having a conditionassociated with mutant-CFTR, said method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of formula (I):

where n R1 is independently chosen from a substituted or unsubstitutedphenyl group, a substituted or unsubstituted heteroaromatic group, or acyclic or acyclic alkyl group; R2 is independently chosen form ahydrogen, a alkyl group, an ether group, a halogen, or a perfluoroalkylgroup; R3 is independently chosen from a hydrogen or an alkyl group, andR4 is independently chosen from a substituted or unsubstitutedheteroaromatic group, or a alkanoyl-amine group; or a pharmaceuticallyacceptable derivative thereof, as an individual stereoisomer or amixture thereof; or a pharmaceutically acceptable salt thereof.
 25. Themethod of claim 24, wherein said condition is cystic fibrosis.
 26. Themethod of claim 24, wherein the subject, after treatment, has a decreasein mucous or bacterial titer in their lungs, a decrease in coughing orwheezing, a decrease in pancreatic insufficiency, or a decrease inelectrolyte levels in their sweat.
 27. The method of claim 24, whereinsaid subject is a non-human animal.
 28. The method of claim 24, whereinthe animal is a mammal.
 29. The method of claim 24, wherein themutant-CFTR is a gating defective mutant-CFTR.
 30. The method of claim29, wherein the gating defective mutant-CFTR is ΔF508-CFTR, G551D-CFTR,G1349D-CFTR, or D1152H-CFTR.
 31. A method of increasing ion permeabilityof a cell producing a mutant-CFTR protein, said method comprisingcontacting said cell with a compound of formula (I):

where n R1 is independently chosen from a substituted or unsubstitutedphenyl group, a substituted or unsubstituted heteroaromatic group, or acyclic or acyclic alkyl group; R2 is independently chosen form ahydrogen, a alkyl group, an ether group, a halogen, or a perfluoroalkylgroup; R3 is independently chosen from a hydrogen or an alkyl group, andR4 is independently chosen from a substituted or unsubstitutedheteroaromatic group, or a alkanoyl-amine group; or a pharmaceuticallyacceptable derivative thereof, as an individual stereoisomer or amixture thereof; or a pharmaceutically acceptable salt thereof, whereinsaid compound is provided in an amount effective to increase ionpermeability of said cell.
 32. The method of claim 31, wherein said cellcontains a recombinant expression cassette that encodes said mutant-CFTRprotein.
 33. The method of claim 31, wherein said cell contains a genomethat encodes said mutant-CFTR protein.
 34. The method of claim 31,wherein said ion permeability increases an ion transporting activitythat increases a rate of transport of ions across the plasma membrane ofsaid cell.
 35. The method of claim 31, wherein the mutant-CFTR is agating defective mutant-CFTR.
 36. The method of claim 35, wherein thegating defective mutant-CFTR is ΔF508-CFTR, G551D-CFTR, G1349D-CFTR, orD1152H-CFTR.
 37. A method of treating a subject having cystic fibrosis,the method comprising: identifying a mutant-CFTR in the subject; andadministering to the subject a pharmaceutical composition comprising acompound of formula (I):

where n R1 is independently chosen from a substituted or unsubstitutedphenyl group, a substituted or unsubstituted heteroaromatic group, or acyclic or acyclic alkyl group; R2 is independently chosen form ahydrogen, a alkyl group, an ether group, a halogen, or a perfluoroalkylgroup; R3 is independently chosen from a hydrogen or an alkyl group, andR4 is independently chosen from a substituted or unsubstitutedheteroaromatic group, or a alkanoyl-amine group; or a pharmaceuticallyacceptable derivative thereof, as an individual stereoisomer or amixture thereof; or a pharmaceutically acceptable salt thereof FTR. 38.The method of claim 37, wherein the gating defective mutant-CFTR is aG551D-CFTR, G1349D-CFTR, or D1152-CFTR.
 39. The method of claim 37,wherein said subject is a non-human animal.
 40. The method of claim 39,wherein the animal is a mammal.